Investigations on the effects of dietary insoluble and ...

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Investigations on the effects of dietary insoluble and soluble non-starch

polysaccharides (NSP) on host-parasite interactions in laying hen chicks

infected with Heterakis gallinarum or Ascaridia galli

Dissertation

zur Erlangung des Doktorgrades

der Fakultät für Agrarwissenschaften

der Georg-August-Universität Göttingen

vorgelegt von

Gürbüz Daş

Geboren in Göle, Türkei

Göttingen, November 2010

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1. Supervisor: Prof. Dr. Dr. Matthias Gauly

1. Co-supervisor: Prof. Dr. Hansjörg Abel

Date of dissertation: 18th November 2010

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Dedicated to

the memories of my mother

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TABLE OF CONTENTS

LIST OF TABLES ...............................................................................................................iii LIST OF FIGURES............................................................................................................... v LIST OF ABBREVIATIONS .............................................................................................. vi SUMMARY .......................................................................................................................... 1 CHAPTER - I ........................................................................................................................ 4

1.1. Foreword..................................................................................................................... 5 1.2. General introduction................................................................................................... 6 1.2.1. Nutrition of host animal and parasitic infections..................................................... 6 1.2.2. Non starch polysaccharides (NSP) ........................................................................ 10 1.2.3. Physiological effects of NSP ................................................................................. 12

1.2.3.1. Insoluble NSP................................................................................................. 12 1.2.3.2. Soluble NSP.................................................................................................... 13 1.2.3.3. Inulin............................................................................................................... 14

References ....................................................................................................................... 14 CHAPTER - II ..................................................................................................................... 22

Non-starch polysaccharides alter interactions between Heterakis gallinarum and Histomonas meleagridis .................................................................................................. 23 Abstract............................................................................................................................ 23 2.1. Introduction .............................................................................................................. 24 2.2. Materials and methods.............................................................................................. 25 2.3. Results ...................................................................................................................... 31 2.4. Discussion................................................................................................................. 39 2.5. Conclusion................................................................................................................ 41 References ....................................................................................................................... 42

CHAPTER - III.................................................................................................................... 46 Effects of dietary non-starch polysaccharides on establishment and fecundity of Heterakis gallinarum in grower layers............................................................................. 47 Abstract............................................................................................................................ 47 3.1. Introduction .............................................................................................................. 48 3.3. Results ...................................................................................................................... 56 3.4. Discussion................................................................................................................. 63 3.5. Conclusion................................................................................................................ 66 References ....................................................................................................................... 67

CHAPTER -IV .................................................................................................................... 72 Effects of dietary non-starch polysaccharides in Ascaridia galli-infected grower layers73 Abstract............................................................................................................................ 73 4.1. Introduction .............................................................................................................. 74 4.2. Material and methods ............................................................................................... 75 4.3. Results ...................................................................................................................... 82 4.4. Discussion................................................................................................................. 88 4.5. Conclusion................................................................................................................ 91 References ....................................................................................................................... 91

CHAPTER - V..................................................................................................................... 97 5. General discussions ..................................................................................................... 98 5.1. Body weight development and feed intake............................................................... 98 5.2. Parasitic infections intensified by the dietary NSP ................................................ 100 5.3. Technical issues in determination of nematode egg excretion ............................... 102 5.4. General conclusions................................................................................................ 102 References ..................................................................................................................... 103

ZUSAMMENFASSUNG .................................................................................................. 106

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ACKNOWLEDGEMENTS .............................................................................................. 109 Curriculum Vitae ............................................................................................................... 111

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LIST OF TABLES

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Chapter-I

Table Basic features of Ascaridia galli and Heterakis gallinarum. 8

Chapter-II

Table 1 Number of birds allocated to the experimental groups. 26

Table 2 Composition and analysis of the experimental diets. 29

Table 3 Interaction of the diets and dimetridazole treatment on average

H. gallinarum worm burdens in dimetridazole treated and

untreated birds

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Table 4 Effects of the diets and the dimetridazole treatment on sex

ratio and average worm length.

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Table 5 Effects of diet on feed intake, body weight (BW), and

feed:gain ratio in the pre-infectional period (1-3 wk).

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Table 6 Effects of the investigated factors on feed consumption, body

weight (BW), and feed:gain ratio (as LSMEANS and SE).

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Chapter-III


Table 2 Effects of diet and H. gallinarum infection on feed

consumption, body weight (BW), and feed:gain ratio

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Table 3 Effect of diet on establishment rate, average number of worms

per bird, sex ratio and length of worms in birds infected with

Heterakis gallinarum (200 eggs/bird)

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Table 4 Effect of diet on the amount of faeces, the excretion of

nematode eggs and the fecundity estimates of worms in birds

infected with Heterakis gallinarum (200 eggs / bird).

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Table 5 Effects of diet and H. gallinarum infection on the size of

certain visceral organs

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Table 6 Effects of diet and H. gallinarum infection on biochemical

characteristics of the caeca.

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Chapter-IV


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Page no

Table 2 Effects of diet and A. galli infection on feed consumption,

body weight development (BW), and feed:gain ratio.

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Table 3 Effect of diet on establishment rate, average number of worms

per bird, sex ratio, length and egg excretion parameters of

worms in birds infected with Ascaridia galli.

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Table 4 Effects of diet and A. galli infection on the size of visceral

organs.

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Table 5 Effects of diet and A. galli infection on biochemical

parameters of caecal content.

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LIST OF FIGURES

Page no

Chapter-I

Figure 1 Host-nutrition and parasite interaction concepts. 7

Figure 2 Example of cell wall materials from oats. 10

Chapter-II

Figure 1 Incidence of H. gallinarum infection without (-) and with (+)

dimetridazole treatment of the birds on different diets.

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Figure 2 Establishment rate (%) of Heterakis gallinarum after a single

dose (200 eggs/bird) inoculation of Histomonas meleagridis

positive eggs in chickens, left untreated (-) or treated (+) with

dimetridazole.

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Figure 3 Average daily feed intake of H. gallinarum infected (+) and

uninfected control (-) groups on different diets, without and

with the dimetridazole treatment.

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Chapter-III

Figure Interaction effect (P=0.014) of diet and infection on the

propionate pool.

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Chapter-IV

Figure Pool size of acetate in the caeca as influenced by interaction

between diet and infection.

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LIST OF ABBREVIATIONS

°C Degree Celsius µl Microliter ADF Acid detergent fibre ADG Average daily weight gain AIC Akaike's Information Criterion BIC Schwarz's Bayesian Criterion BW Body weight BWG Body weight gain CaCo3 Calcium carbonate CON Control basal diet CP Crude protein d Day DM Dry matter EE Ether extract

EPD Eggs per day; total number of eggs excreted per worm population of a bird within 24 h

EPD/female EPD based female worm fecundity; number of eggs excreted per female worm within 24 h

EPG Eggs per gram of faeces

EPG / female EPG based female worm fecundity; average number of eggs excreted per female worm through one gram of faeces

FID Flame ionization detector FMVO Futtermittelverordnung (German Feed Regulations) g Gram g Acceleration of gravity, g-force GC Gas chromatography Hs-Index Hepato-somatic index I-NSP Insoluble non-starch Polysaccharide K2Cr2O7 Potassium dichromate LSL Lohmann Selected Leghorn MCP Monocalcium Phosphate ME Metabolizable energy N Total number of observations n number of observations per group or treatment NDF Neutral detergent fibre NSP Non-starch polysaccharide OM Organic matter p. i. post infection SCFA Short chain fatty acids S-NSP Soluble non-starch polysaccharide v/v volume / volume VFA Volatile fatty acids w/v Weight / volume

Summary

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SUMMARY

The objective of this study was to test the hypothesis that low or highly soluble dietary non

starch polysaccharides (NSP) differently affect infections with Heterakis gallinarum or

Ascaridia galli in grower layers. Because H. gallinarum acts as a vector for transmission of

Histomonas meleagridis, the agent of ‘Blackhead disease’, effects of NSP-supplemented

diets on interactions between these two parasites were also investigated.

The experiments were conducted between 2007 and 2010 at the Department of

Animal Sciences of the Göttingen University. Three experimental diets were used: basal

diet (CON), supplying metabolizable energy and nutrients for grower layers according to

recommended feeding standards. One hundred gram of pea bran or chicory root meal were

added to each kg of CON in diets I-NSP and S-NSP supplying insoluble (I-) or soluble (S-)

NSP, respectively. The first study aimed at investigating effects of NSP supplemented diets

on interactions between H. gallinarum and H. meleagridis, including a prophylactic

treatment with dimetridazole (0.05%, w/v) via drinking water against H. meleagridis for

half of the birds. Histomonas free H. gallinarum female worms obtained from this study

were used for the preparation of infection material applied in the experiment that

investigated effects of NSP-supplemented diets on H. gallinarum infection. Effects of

dietary NSP on either H. gallinarum- or A. gall-infections were separately investigated in

two consecutive experiments, each comprising three identical runs for each nematode

species. In each run, three feeding groups of one-day-old female layer chicks were built,

each being fed until an age of wk 3 with one of the three experimental diets. At the end of

wk 3, the birds were marked with wing tags and weighed. Each feeding group was

subdivided into an uninfected control and an infected group of birds, the latter being

inoculated with 200 embryonated eggs of H. gallinarum or 250 embryonated eggs of A.

galli, respectively. Daily feed consumption was determined per group throughout the

experiment until wk 11. In the last two runs of the H. gallinarum-experiment, the infected

birds were placed into individual cages and their daily total amounts of faeces, number of

eggs per gram of faeces (EPG) and total number of eggs excreted within 24 h (eggs per

day, EPD) were determined. In the A. galli-experiment, the faeces were collected at the

time of slaughter and EPG was determined. The birds were slaughtered 8 wk post infection

(p.i.) and their worm burdens were determined. Volatile fatty acids (VFA) and pH were

measured in caeca contents.

Summary

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In the first experiment that dealt with histomonas contaminated H. gallinarum

(Chapter-II), treatment against H. meleagridis significantly increased the incidence of H.

gallinarum infection and the average worm length in all infected groups irrespective of the

type of experimental diet consumed by the birds. An interaction between effects of diet and

dimetridazole treatment indicated that S-NSP resulted in lowest worm burden in

dimetridazole-untreated birds, whereas it caused highest worm burden in treated birds.

Within each feeding group, higher worm burdens were determined in treated than in

untreated birds. Infection with H. gallinarum reduced the body weight (BW) of the chicks,

and H. meleagridis aggravated this effect. Dimetridazole-treated and untreated uninfected

birds developed similar BW. Both NSP-supplemented diets, with S-NSP being inferior to

I-NSP, led to lower BW of the birds.

In the histomonas free H. gallinarum-experiment (Chapter-III), the NSP-

supplemented diets elevated the incidence of infection, the average number of larvae and

the total worm burden compared to CON. The worm length was not influenced by the type

of diet. The daily amount of faeces increased in NSP-fed birds. The EPG, EPD and female

worm fecundity (EPD/female worm) were elevated after feeding S-NSP, whereas I-NSP

led to lower EPG/female worm. The EPD increased in the sequence of CON < I-NSP < S-

NSP. Both, the NSP-supplemented diets and infection led to reduced BW of birds and

infection additionally impaired the feed conversion rate. The NSP-supplemented diets

increased average length of caecum with S-NSP exerting a stronger effect than I-NSP.

Filled caeca weight was increased by S-NSP. The infection increased the weight of filled

and emptied (washed) caeca. Feeding S-NSP lowered intracaecal pH and molar proportion

of acetate and increased that of butyrate compared to CON and I-NSP. Caecal pool of VFA

was increased with S-NSP. Infection increased intracaecal pH, accompanied by lower

molar proportion of butyrate and reduced caecal pools of VFA.

In the A. galli-experiment (Chapter-IV) both NSP-diets, particularly S-NSP,

increased incidence of infection and worm burden of the birds, but the development

(length) and fecundity of the nematode remained unaffected. A. galli-infection caused a

less efficient feed utilization for body weight gain (BWG) resulting in lower BW

irrespective of type of diet consumed. NSP-fed birds, particularly those on S-NSP, showed

retarded BW development compared to birds receiving CON. Intracaecal pH was lowered

by feeding S-NSP but was unaffected by A. galli-infection. Both NSP-diets increased

caecal VFA pool size, S-NSP exerting a greater effect than I-NSP. Infected birds had

smaller caecal VFA pool size than their uninfected counterparts consuming the

Summary

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corresponding diets. S-NSP also led to higher weights of splanchnic tissues and intestinal

tract. These effects were less pronounced in I-NSP fed chicken.

The results show that H. meleagridis does not only harm the definitive host, but

also its vector, H. gallinarum. Both, insoluble and soluble dietary NSP favor H. gallinarum

infection while S-NSP additionally intensifies histomonas-infection, which then impairs

establishment and development of H. gallinarum. The pea bran and chicory root meal used

as sources of insoluble and soluble dietary NSP, respectively, favored the establishment of

histomonas-free H. gallinarum in grower layers. Inulin rich chicory root meal additionally

enhanced fecundity of this nematode. Insoluble and soluble dietary NSP retard growth

performance, alter gastrointestinal environment and lead to higher weights of splanchnic

tissues associated with an elevated establishment of A. galli in grower layers. The NSP

supplemented diets, and S-NSP in particular, may have altered the gastrointestinal

environment, which in turn enhanced nematode infections. It is concluded that the two

natural sources of insoluble and soluble NSP offer no potential as protecting agents against

the parasitic infections in chicken. Therefore, suitable measures of precaution should be

applied to production systems particularly prone to gastrointestinal parasitic infections and

where diets with relatively high NSP-contents are fed.

Chapter-I

Background

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CHAPTER - I

BACKGROUND

Chapter-I

Background

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1.1. Foreword

As a consequence of recent changes in consumer demands toward chemical-

residues free animal products and increasing public concern about animal welfare,

conventional cage production systems for laying hens are being replaced by outdoor/free-

range systems in the EU (Gauly et al., 2001; Permin and Ranvig, 2001). The proportion of

laying hen farms in the form of non-cage production systems increased from 6.7 % in 2000

to 33.8% in 2007, and will be 100% by 2010 in Germany (ZMP, 2008). In some countries,

e.g. in Switzerland the battery cages have completely been banned (Kaufmann-Bart and

Hoop, 2009), and in Austria, almost 70% of the laying hens are kept in the non-cage

production systems (ZMP, 2008). These changes reflect a transition period that is

determined by an EU-wide ban on the use of battery cages (un-enriched cages) for laying

hens, and will enter into force in January 2012 (Anonymous, 1999). Changes that resulted

from increased numbers of chickens kept in outdoor-floor husbandry systems have caused

re-emerging parasitic infections (Permin et al., 1999; Thamsborg et al., 1999; Fossum et

al., 2009; Kaufmann and Gauly, 2009). The prevalence of nematode infections in battery

cage systems was low (< 5%; Permin et al., 1999). However, because of the faeces

management that allows nematodes to complete their life cycles, and the frequent contact

of animals with faeces, there is an increased risk for ingestion of parasitic stages in floor-

outdoor access husbandry systems. Among the parasitic infections, the most prevalent

infections are with Heterakis gallinarum and Ascaridia galli (Permin et al., 1999;

Kaufmann and Gauly, 2009). These two parasites are probably the most important

nematode species of economical importance in chickens.

Fibre rich diets for poultry are expected to increase in the future, particularly in

organic poultry production (Sundrum et al., 2005; Van de Weerd et al., 2009). In addition,

poultry housed in floor systems can ingest fibre rich litter material from the floor.

Similarly, layers in modified cages can also pick up fibrous material from the litter bath

(Hetland et al., 2004). In free-range systems, birds can directly consume plants available in

the outdoor area. Non starch polysaccharides (NSP) are the major part of dietary fibre.

Monogastric animals do not possess own digestive enzymes for NSP degradation.

Depending on its fermentability, this class of carbohydrates can either less or highly be

utilized by the microorganisms in the distal intestinal tract (Englyst, 1989; Bach Knudsen,

2001). There is evidence that dietary NSP may interact with parasites of the host animals.

Feeding grower layers with NSP that form viscous digesta, favored the development of A.

Chapter-I

Background

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galli (Daenicke et al., 2009). In pigs, the type of dietary NSP has been shown to affect the

establishment, development and fecundity of pig-specific common nematodes differently

(Petkevičius et al., 1997; 2001; 2003). The main objective of the present study was to

investigate the effects of less or highly fermentable dietary NSP on H. gallinarum and A.

galli infections in growing layer hens. Because of the vector role of H. gallinarum in the

transmission of Histomonas meleagridis, the effects of NSP supplemented diets on

interactions between these two parasites were also investigated.

1.2. General introduction

1.2.1. Nutrition of host animal and parasitic infections

Feed is probably the most important entity in poultry production that can expose the birds

to a wide variety of factors through the gastrointestinal tract (Yegani and Korver, 2008).

Dietary characteristics can modulate a bird’s susceptibility to infectious challenges and

subtle influences due to the level of nutrients, or the types of ingredients may at times be of

critical importance (Klasing, 1998). There exists a large body of evidence that the host

animal nutrition can alter the interactions between the host animals and their parasites

(Coop and Holmes, 1996; Coop and Kyriazakis, 1999; Stear et al., 2007). Nutrition can

affect resistance and/or resilience status of host animals through specific adaptive

physiological responses and/or certain immune regulations (Figure 1).

Interactions between the host and nutrition can be considered from two interrelated

perspectives. Firstly, the effects of nutrition on the metabolic disturbances and

pathophysiology induced by parasitism, and secondly the influence of nutrient availability

on the ability of the host to mount an effective response against parasite establishment

and/or development and to induce parasite rejection. The level of nutrition can thus

influence the ‘resilience’ and resistance’ of the host to parasitic infections (Coop and

Kyriazakis, 1999). Nutrition can affect gastrointestinal nematodes through its influence on

resistance, i.e. the ability to regulate gastrointestinal nematode establishment, fecundity

and survival. This is mainly mediated through acquired immunity, and thus nutrition has

the potential to affect the rate of acquisition and/or the degree of expressing of immunity

(Kyriazakis and Houjdijk, 2006).

Chapter-I

Background

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Figure 1. Host-nutrition and parasite interaction concepts (modified after Coop and

Kyriazakis, 1999; Hoste, 2001; Kyriazakis and Houjdijk, 2006).

Resilience can be considered as the host’s ability to maintain a reasonable level of

productivity in the face of a parasitic challenge (Coop and Kyriazakis, 1999). In other

words, resilience is the ability of the host to maintain its physiological functions and to

tolerate the detrimental effects due to parasites (Hoste, 2001). Further common terms

describing interactions between host and parasitic infections are susceptibility and

tolerance. Susceptibility is the opposite of resistance. Tolerance is similar to resilience, and

refers to the ability of a host to perform despite the presence of infection. Resilience is

preferred over tolerance, because it is used to avoid the confusion with immunological

tolerance (Abdelqader, 2007). Effects of protein supplementation on resistance

and/resilience of gastrointestinal nematodes of ruminants are well known (Stear et al.,

2007). Wallace et al. (1995) showed that protein supplementation did not influence worm

burden of lambs infected with a blood sucking nematode, Haemonchus contortus, but

lowered faecal egg counts and increased packed red cell volume. Dietary supplementation

with urea also enhanced resistance and resilience to Trichostrongylus colubriformis (Knox

and Steel, 1999).

Certain dietary components can directly influence gastrointestinal parasites through

their antiparasitic compounds. Various secondary plant metabolites, e.g. phenolic

metabolites, nitrogen containing metabolites and terpenoids, are thought to have

Host

Resilience (Physiological adaptations)

Resistance (Immune regulations)

Acquisition

Reversal of metabolic

disturbances

Reversal of pathophysiology

Expression

Nutrition

Chapter-I

Background

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antiparasitic properties (Coop and Kyriazakis, 2001). Plants rich in tannins, a class of

phenolic secondary metabolites, are known to have detrimental effects on gastrointestinal

parasitic infections of ruminants (Hoste et al., 2006).

It appears that effects of nutrition on gastrointestinal nematodes have extensively

been examined in ruminants. However, there is evidence that nutrition can influence

poultry parasites too. Among the parasitic infections, the most prevalent infections are with

Heterakis gallinarum and Ascaridia galli (Permin et al., 1999; Kaufmann and Gauly,

2009). These two parasites are probably the most important nematode species of

economical importance in chickens. It has been reported that vitamins (Idi et al., 2007),

minerals (Gabrashanska et al, 2004) protein or amino acids (Riedel and Ackert, 1951; Daş

et al., 2010) alter infections of poultry with A. galli. Compared to A. galli, less is known

about dietary effects on the caecal worm, Heterakis gallinarum. Basic features of the two

nematodes are summarized in the following table.

Table. Basic features of Ascaridia galli and Heterakis gallinarum.

A. galli H. gallinarum

Life cycle Direct Direct

Infective stage L3 L3

Histotrophic phase + + (?)

Prepatent period 4-8 wk 24 d

Adult length, cm

Female 6.0 - 12.0 1.0 -1.5

Male 5.0 - 7.8 0.7-1.3

Predilection site small intestine ceca

Feeding on Digesta Digesta / bacteria

Both nematodes have direct life cycles, i.e. require no intermediate host to transmit

to their definitive hosts (Herd and McNaught, 1975; McDougald, 2005). Infection starts

with ingestion of infective larval stages (L3) in form of embryonated eggs by the host

animal. Embryonated eggs of A. galli containing L3 larva hatch in the proventriculus or

duodenum within 24 h after ingestion (Idi, 2004). The larva invades the mucosal layer of

the intestine, where a histotropic phase takes place. The histotropic phase is a normal part

of the life cycle of A. galli and it lasts approximately 7 to 50 days, depending on infection

dose (Herd and McNaught, 1975). The pathogenicity of A. galli is considered to be

stronger during histotropic, larval development, resulting in inflammation and injury to the

Chapter-I

Background

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intestinal wall and to the host's absorption of metabolic waste from the nematode

(Ramadan and Abou Znada, 1991). It is not clear whether life cycle of H. gallinarum

involves a histotropic phase. However, according to Van Grembergen (1954), Hsü et al.

(1940) have shown the phenomena for H. gallinarum. Prepatent period, the time required

from ingestion of L3 larvae until mature adult parasites are producing eggs, for A. galli is

between 4-8 wk (Idi, 2004; Ramadan and Abou Znada, 1992). H. gallinarum has an

average prepatent period of 24 d, however, it was shown that the females can produce eggs

as early as 21 d after infection (Fine, 1975).

A. galli is the largest nematode parasite of poultry. The length of adults varies

between 5 to 7.76 cm in males and 6 to 11.6 cm in females, respectively (Idi, 2004;

Ramadan and Abou Znada, 1992). A. galli resides mainly in the upper part of the small

intestine, but also can be found in the distal parts, i.e. ileum. A. galli infection can

influence digestion and absorption of nutrients (Hurwitz et al., 1972ab; Walker and Farrell,

1976). A. galli not only retards performance but can also threaten the general intestinal

health of the birds. Dahl et al. (2002) reported that chickens infected with A. galli are at

higher risk of being subjected to outbreaks of fowl cholera with P. multocida.

H. gallinarum has a narrow predilection site, i.e. the caeca, and is regarded as a

relatively less pathogenic nematode (Taylor et al., 2007). However, the importance of this

nematode lies in its role as a main vector for the transmission of Histomonas meleagridis,

the causative agent of ‘blackhead’ disease (McDougald, 2005). Susceptibility of turkeys to

histomonas infection is higher than that of chickens. However, histomonioasis outbreaks

can increase flock mortality and decrease egg production in laying hens (Esquenet et al.,

2003).

Gastrointestinal bacterial flora seems to play important roles in establishment of

both A. galli and H. gallinarum. Johnson and Reid (1973) showed that lower number of A.

galli larvae established themselves in germ-free chickens than in chickens with

conventional flora. Chickens inoculated with single species of bacteria harbored higher

number of larvae than germ-free birds, but had lower number of larvae than those with a

conventional flora. Although establishment of A. galli is enhanced by the presence of

bacteria, it was shown that germ-free birds harbor established larvae (Johnson and Reid,

1973). For H. gallinarum, the role of bacteria seems to be more vital (McDougald, 2005).

Springer et al. (1970) showed that Heterakis larvae were not able to survive when injected

into caeca of gnotobiotic birds. Moreover, H. gallinarum is regarded as a bacteria feeder

(Bilgrami and Gaugler, 2004). H. meleagridis attains full virulence in the caeca of chicken

Chapter-I

Background

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only in combination with the presence of several bacteria species (Springer et al., 1970;

McDougald, 2005). Because both A. galli and H. gallinarum reside in the gastrointestinal

tract, it is likely that an altered gastrointestinal environment due to dietary characteristics

may influence their establishment and fecundity. Moreover, dietary characteristics may

aslo alter interactions between bacteria dependent H. gallinarum and H. meleagridis.

1.2.2. Non starch polysaccharides (NSP)

Today’s poultry diets consist of highly concentrated feedstuffs providing efficient

digestion and utilization. The diets are mainly based on cereals and protein rich

ingredients. Fibre is rather regarded as nutrient diluent or anti-nutrient, depending on its

solubility. Cereals and legumes, the bulk of commercial poultry diets, contain a significant

amount of fibre (Hetland et al., 2004). Plant polysaccharides can be separated broadly into

two distinct and chemically well-defined types; the storage polysaccharide starch (α-

glucan) and the cell-wall polysaccharides (non- α-glucan), which may conventionally be

called non starch polysaccharides (NSP). The term dietary fibre is used for the sum of NSP

and lignin (Bach Knudsen, 2001). A typical arrangement of cereal polysaccharides is

illustrated in the following figure.

Figure 2. Example of cell wall materials from oats (Bach Knudsen, 2001).

Chapter-I

Background

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As shown in the figure, plant polysaccharides consist of the storage polysaccharide

starch and the cell wall polysaccharides. Starch is composed of amylose and amylopectin,

which contain α-(1-4) and α-(1-6)-glucosidic linkages, respectively (Montagne et al.,

2003). The building blocks of the cell wall polysaccharides are the pentoses arabinose and

xylose, the hexoses glucose, galactose and mannose, the 6-deoxyhexoses rhamnose and

fucose, and the uronic acids glucuronic and galacturonic acid (Bach Knudsen, 2001).

Another major component of the cell wall is lignin which can be described as very

branched networks built up by phenylpropane units. Lignin cements and anchors the

cellulose microfibrils and other matrix polysaccharides (Bach Knudsen, 2001). The main

polysaccharides of plant cell walls are cellulose, pectins, β-glucans [mixed linked β (1→3)

(1→4)-D-glucan (β-glucan)], pentosans, xylans (Montagne et al., 2003; Bach Knudsen,

2001).

Dietary starch can be hydrolyzed by pancreatic α-amylase and may therefore be

digested in the small intestine and be absorbed as glucose (Englyst, 1989). It is generally

accepted that starch is well digested in the gastrointestinal tract (Classen, 1996; Józefiak et

al., 2004). In contrast to starch, NSP are not susceptible to the endogenous enzymes and,

depending on their fermentability can either less or highly be utilized by the

microorganisms in the distal parts of the gastrointestinal tract (Englyst, 1989; Schneeman,

1999; Montagne et al., 2003). Cellulose and xylans belong to insoluble NSP, whereas

pectins, β-glucans and arabinoxylans are considered as soluble NSP (Hetland et al., 2004).

Plants generally contain a mixture of soluble and insoluble NSP in a ratio that varies

between plants, plant parts, and stage of maturity (Montagne et al., 2003; Hetland et al.,

2004).

Inulin and oligofructose are comparable to dietary fibre in that they are composed

of multiple saccharide units, which are soluble in water and are not digested by the

endogenous enzymes found in the intestines (Schneeman, 1999). Chemically, inulin fructo-

oligosaccharides (FOS) are composed of linear chains of fructose units, linked by β-(2→1)

fructosyl-fructose bonds, often terminated by a glucose unit (Ten Bruggencate et al., 2004;

Roberfroid, 2005). The number of fructosyl moieties ranges from 2 to 60 for inulin and

from 2 to 7 for FOS. In vitro, fermentation experiments revealed that molecules with a

degree of polymerization (DP) > 10 are fermented, on average, half as quickly as

molecules with a DP of < 10 (Ten Bruggencate et al., 2004; Rehman et al., 2008). The only

plant that has so far been used industrially for the extraction of inulin-type fructans belongs

to the Compositae family, i.e. chicory (Roberfroid, 2005).

Chapter-I

Background

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1.2.3. Physiological effects of NSP

The main physico-chemical properties of dietary fibre with nutritional significance are the

cation exchange capacity, hydration properties, viscosity and organic compound absorptive

properties (Bach Knudsen, 2001). However, hydration and viscosity associated properties

of dietary fibre appear to have been studied most extensively in poultry nutrition (Hetland

et al., 2004). The hydration properties are characterized by the swelling capacity, solubility

and water holding capacity, and are linked to the type of polymers that make up the cell

wall and their intermolecular association. Water holding capacity is also used to describe

hydration properties and reflects the ability of a fibre source to incorporate water within its

matrix. In general cereal fibre tends to have lower water holding capacity than fibre

sources high in pectin containing materials. The majority of polysaccharides give viscous

solutions when dissolved in water. The viscosity is primarily dependent on the molecular

weight of the polymer and the concentration. Large molecules increase viscosity of diluted

solutions and their ability to do this mainly depends on the volume they occupy. The

volume of the polymers is much greater than that of monomers and the volume occupied

by one polymer coil will be greater than the combined volume of two coils each half its

length (Bach Knudsen, 2001). Because of the importance of the complex interactions

between different chemical components of plant tissues, it makes little sense from a

nutritional point of view, to describe dietary fibre solely in chemical terms. Rather, it may

be better to describe the cell wall polysaccharide components of feedstuffs in terms of their

physicochemical properties, which are likely to be related to their physiological effects

(Smits and Annison, 1996).

1.2.3.1. Insoluble NSP

The insoluble fibre fraction has traditionally been regarded as a nutrient diluent in

monogastric animal diets (Hetland et al., 2004). Insoluble polysaccharides such as

cellulose and xylans can hold water as they behave like sponges but their viscosity

properties are relatively unimportant (Smits and Annison, 1996). In contrast to soluble

fibre, insoluble fibre is not extensively degraded by bacterial fermentation in poultry,

which makes its influence on the composition and quantity of the microflora relatively

insignificant (Hetland et al., 2004). Therefore, unlike soluble fibre, insoluble fibre does

merely influence the composition and population size of the gastrointestinal microflora

Chapter-I

Background

13

13

(Shakouri et al., 2006). The most obvious effect of insoluble NSP is the increased bulk of

digesta in the intestinal tract. This may be handled either by a larger capacity of the

digestive system or a faster passage rate through the tract (Hetland et al., 2004). Inclusions

of insoluble NSP in poultry diets have certain positive effects on animal welfare. Van

Krimpen et al. (2007; 2008) reported that hens fed diets high in insoluble NSP increased

time spent for eating and reduced aggressive pecking behaviours. Diets supplemented with

NSP may also stimulate the development of the gizzard suggesting improved digestive

functioning (Van Krimpen et al., 2009).

1.2.3.2. Soluble NSP

It is well known that soluble NSP exert anti-nutritive effects in growing poultry through

viscosity associated effects (Choct and Annison, 1992; Daenicke et al., 1999; Francesch

and Brufau, 2004; Daenicke et al., 2009). Soluble fibres can produce high viscosity in the

small intestine and thereby inhibit digestion and absorption. High viscosity can affect feed

intake due to slower digesta passage rate, which in turn causes microbial proliferation in

the intestine (Van der Klis et al., 1993; Hetland et al., 2004; Yegani and Korver, 2008).

The water holding capacity of soluble fibre is associated with sticky droppings (Hetland et

al., 2004). Due to their NSP contents, barley, wheat, rye and oats can increase viscosity,

decrease digesta passage rate, digestive enzymatic activities and nutrient digestibility,

which may consequently cause depressed feed conversion efficiency and growth rate of

birds (Yegani and Korver, 2008). The viscous properties of NSP can impair the diffusion

and convective transport of lipase, oils and bile salt micelles within the gastrointestinal

tract. NSP induced increased viscosity may stimulate mucus secretion by the Goblet cells.

Morever, increased viscosity may reduce the contact between potential nutrients (e.g. fats)

and the digestive secretions (e.g. lipases, bile salts), and impair the transport to the

epithelial surface (Smits and Annison, 1996). NSP-caused high digesta viscosity is often

associated with increased gastrointestinal capacity. Iji et al. (2001) reported that the gross

weight of the intestines as well as the mucosal morphometry of the small intestine was

increased by NSP of highly viscous properties. Viscosity associated effects of soluble

NSP are not limited to impaired digestion and absorption of nutrients, but are also closely

related to the microbial proliferation in the gastrointestinal tract. Increased average digesta

retention time due to higher viscosity, likely provides favorable conditions for bacterial

proliferation and activity. Because most of the soluble NSP are fermentable, local or

Chapter-I

Background

14

14

systemic effects of pathogenic bacteria may threaten general health of birds (Smits and

Annison, 1996; Shakouri et al., 2006; Yegani and Korver, 2008). Viscosity associated anti-

nutritive effects of soluble NSP can to some extent be eliminated through exogenous NSP

degrading enzymes supplemented to the diets (Castanon et al., 1997; Dusel et al., 1998;

Mikulski et al., 2006; Józefiak, et al., 2007; Daenicke et al., 2009).

1.2.3.3. Inulin

Unlike NSP with high viscosity properties, inulin does not appear to increase intestinal

viscosity (Schneeman, 1999). It can act as a prebiotic, i.e., it may be a selectively

fermented ingredient that allows specific changes in the composition or activity of the

gastrointestinal microbiota (Rehman et al., 2008). Due to its β-(2→1) linkages, it is

resistant to enzymatic hydrolysis in the upper gastrointestinal tract and reaches intact to

distal parts of the tract, where it is completely fermented (Juskiewicz and Zdunczyk, 2004).

End products of inulin fermentation are short chain fatty acids (SCFA), carbon dioxide,

methane and hydrogen (Donalson et al., 2008). Although fermentation of inulin may start

already in the ileum, caeca are the main site of microbial fermentation in chickens

(Juskiewicz et al., 2005). An inulin-dependent stimulation of metabolic activity of

beneficial intestinal bacteria has been reported for turkeys (Juskiewicz et al., 2005) and

chickens (Rehman et al., 2007). Although many reports suggest that inulin stimulated

bacteria may inhibit colonisation of intestinal pathogens resulting in a fermentation

benefical to the health of the animals (Juskiewicz et al., 2005; Rehman et al., 2007;

Donalson et al., 2008), there are exceptions. Ten Bruggencate et al. (2004) showed that

inulin and fructo-oligosaccharides impaired resistance to salmonella infections in rats.

According to these authors this might be due to rapid production of fermentation

metabolites and subsequent impairment of the mucosal barrier.

References

Abdelqader, A.M.A., 2007. Characterization of local chicken and their production systems

in Jordan with comparative studies on parasitological infections. Ph.D. Thesis. p:27.

University of Göttingen, pp:127.

Chapter-I

Background

15

15

Anonymous, 1999. Official Journal of the European Communities. COUNCIL

DIRECTIVE 1999/74/EC laying down minimum standards for the protection of

laying hens. Official Journal of the European Communities, L 203/ 53.

Bach Knudsen, K.E., 2001. The nutritional significance of “dietary fibre” analysis. Anim.

Feed Sci. Technol. 90, 3-20.

Bilgrami, A.L., Gaugler, R., 2004. Feeding behaviour. In: Gaugler, R., Bilgrami, A.L.

(Eds.), Nematode Behaviour. CABI publishing, pp. 98.

Castanon, J.I.R., Flores, M.P., Petterson, D., 1997. Mode of degradation of non-starch

polysaccharides by feed enzyme preparations. Anim. Feed. Sci. Technol. 68, 361-

365.

Choct, M., Annison, G., 1992. Anti-nutritive effect of wheat pentosans in broiler chickens:

roles of viscosity and gut microflora.

Classen, H.L., 1996. Cereal grain starch and exogenous enzymes in poultry diets. Anim.

Feed Sci. Technol. 62, 21-27.

Coop, R.L., Holmes, P.H., 1996. Nutrition and parasite interaction, Int. J. Parasitol.

26,951-962.

Coop, R.L., Kyriazakis, I., 1999. Nutrition-parasite interaction. Vet. Parasitol. 84, 187-204.

Coop, R.L., Kyriazakis, I., 2001. Influence of host nutrition on the development and

consequences of nematode parasitism in ruminants. Trends Parasitol. 17, 325-330.

Daenicke, S., Dusel, G., Jeroch, H., Kluge, H., 1999. Factors affecting efficiency of NSP-

degrading enzymes in rations for pigs and poultry. Agribiol. Res, 52, 1-24.

Daenicke, S., Moors, E., Beineke, A., Gauly, M., 2009. Ascaridia galli infection of pullets

and intestinal viscosity: consequences for nutrient retention and gut morphology.

Br. Poult. Sci. 50, 512-520.

Dahl, C., Permin, A., Christensen, J.P., Bisgaard, M., Muhairwa, A.P., Petersen, K.M.D.,

Poulsen, J.S.D., Jensen, A.L., 2002. The effect of concurrent infections with

Pasteurella multocida and Ascaridia galli on free range chickens. Vet. Microbiol. 86,

313-324.

Daş, G., Kaufmann, F., Abel, H., Gauly, M., 2010.Effect of extra dietary lysine in

Ascaridia galli-infected grower layers. Vet. Parasitol. 170, 238-243.

Chapter-I

Background

16

16

Donalson, L. M., Kim, W. K., Chalova, V. I., Herrera, P., McReynolds, J. L., Gotcheva,

V. G., Vidanovic, D., Woodward, C. L., Kubena, L. F., Nisbet, D. J., Ricke, S.C.,

2008. In vitro fermentation response of laying hen cecal bacteria to combinations of

fructooligosaccharide prebiotics with alfalfa or a layer ration. Poultry Sci. 87, 1263-

1275.

Dusel, G., Kluge, H., Jeroch, H., 1998. Xylanase supplementation of wheat-based rations

for broilers: influence of wheat characteristics. J. Appl. Poultry Res. 7, 119-131.

Englyst, H., 1989. Classification and measurement of plant polysaccharides. Anim. Feed

Sci. Technol. 23, 27-42.

Esquenet, C., De Herdt, P., Bosschere, H., Ronsmans, S., Ducatelle, R., Van Erum, J.,

2003. An outbreak of histomoniasis in free-range layer hens. Avian Pathol. 32, 305-

308.

Fossum, O., Jansson, D.S., Etterlin, P.E., Vågsholm, I., 2009. Causes of mortality in laying

hens in different housing systems in 2001 to 2004. Acta Vet. Scand. 51, Artn: 3.

doi:10.1186/1751-0147-51-3

Francesch, M., Brufau, J., 2004. Nutritional factors affecting excreta/litter moisture and

quality. World. Poult. Sci. J. 60, 64-75.

Gabrashanska, M., Teodorova, S.E., Galvez-Morros, M.M., Tsocheva-Gaytandzhieva, N.,

Mitov, M., Ermidou-Pollet, S., Pollet, S., 2004. Administration of Zn-Co-Mn basic

salt to chickens with ascaridiosis II. Sex ratio and microelement levels in Ascaridia

galli and in treated and untreated chickens. Parasitol. Res. 93, 242-247.

Gauly, M., Bauer, C., Mertens, C., Erhardt, G., 2001. Effect and repeatability of Ascaridia

galli egg output in cockerels following a single low dose infection. Vet. Parasitol. 96,

301-307.

Herd, R.P., Mcnaught, D.J., 1975. Arrested development and the histotropic phase of

Ascaridia galli in the chicken. Int. J. Parasitol. 5, 401-406.

Hetland, H., M. Choct, and B. Svihus. 2004. Role of insoluble non-starch polysaccharides

in poultry nutrition. World. Poult. Sci. J. 60, 415–422.

Hoste, H., 2001. Adaptive physiological processes in the host during gastrointestinal

parasitism. Int. J. Parasitol. 31, 31-244.

Chapter-I

Background

17

17

Hoste, H., Jackson, F., Athanasiadou, S., Thamsborg, M.S., Hoskin, S.O., 2006. The

effects of tannin-rich plants on parasitic nematodes in ruminants. Trends Parasitol.

22, 253-261.

Hsü, H.F., Li, S.Y., 1940. Chin. Med. J. 57, 559.

Hurwitz, S., Shamir, N., Bar, A., 1972 (a). Effect of Ascaridia galli on lumen activity of

enzymes in the intestine of chicks. Poultry Sci. 51, 1462-1463.

Hurwitz, S., Shamir, N., Bar, A., 1972 (b). Protein digestion and absorption in the chick:

effect of Ascaridia galli. Am. J. Clin. Nutr. 25, 311-316.

Idi, A., 2004. Effect of selected micronutrients and diets on the establishment and

pathogenicity of Ascaridia galli in chickens. Ph.D. thesis, pp:21. The Royal

Veterinary and Agricultural University, Copenhagen, Denmark.

Idi, A., Permin, A., Jensen, S.K., Murrel, K.D., 2007. Effect of a minor vitamin A

deficiency on the course of infection with A. galli (Schrank, 1788) and the

resistance of chickens. Helminthologia, 44, 3-9.

Iji, P.A., Saki, A.A., Tivey, D.R., 2001. Intestinal development and body growth of broiler

chicks on diets supplemented with non-starch polysaccharides. Anim. Feed Sci.

Technol. 89, 175-188.

Johnson, J., Reid, W.M., 1973. Ascaridia galli (Nematoda): Development and survival in

gnotobiotic chickens. Exp. Parasitol. 33, 95-99.

Józefiak, D., Rutkowski, A. Jensen, B.B., Engberg, R.M., 2007. Effects of dietary

inclusion of triticale, rye and wheat and xylanase supplementation on growth

performance of broiler chickens and fermentation in the gastrointestinal tract.

Anim. Feed Sci. Technol. 132, 79-83.

Józefiak, D., Rutkowski, A., Martin, S.A., 2004. Carbohydrate fermentation in the avian

ceca: a review. Anim. Feed Sci. Technol. 113, 1-15.

Juskiewicz, J., Jankowski, J., Zdunczyk, Z., Biedrzycka, E.l., Koncicki, A., 2005.

Performance and microbial status of turkeys fed diets containing different levels of

inulin. Arch. Gefluegelkd. 69, 175-180.

Juskiewicz, J., Zdunczyk, Z., 2004. Effects of cellulose, carboxymethylcellulose and inulin

fed to rats as single supplements or in combinations on their caecal parameters.

Comp. Biochem. Phys. A. 139, 513-519.

Chapter-I

Background

18

18

Kaufmann, F., Gauly, M., 2009. Prevalence and burden of helminths in laying hens kept in

free range systems. Proceedings of the XIV International Congress for Animal

Hygiene, Vol. 2: 557-559. Vechta, Germany.

Kaufmann-Bart, M., Hoop, R.K., 2009. Diseases in chicks and laying hens during the first

12 years after battery cages were banned in Switzerland. Vet. Rec. 164, 203-207.

Klasing, K.C., 1998. Nutritional modulation of resistance to infectious diseases. Poultry

Sci. 77, 1119-1125.

Knox, M.R., Steel, J.W., 1999. The effects of urea supplementation on production and

parasitological responses of sheep infected with Haemonchus contortus and

Trichostrongylus colubriformis. Vet. Parasitol. 83, 123-135.

Kyriazakis, I., Houdijk, J., 2006. Immunonutrition: Nutritional control of parasites. Small

Ruminant Res. 62, 79-82.

McDougald, L.R., 2005. Blackhead disease (Histomoniasis) in poultry: A critical review.

Avian Dis. 49, 462-476.

Mikulski, D., Jankowski, J., Zdunczyk, Z., Juskiewicz, J., Klebukowska, L., Mikulska, M.,

2006. Performance and gastrointestinal responses of turkeys to different levels of

enzyme (xylanase and glucanase) in a diet. Medycna Wet. 62, 887-892.

Montagne, L., Pluske, J.R., Hampson, D.J., 2003. A review of interactions between dietary

fibre and the intestinal mucosa, and their consequences on digestive health in young

non-ruminant animals. Anim. Feed Sci. Technol, 108, 95-117.

Permin, A., Bisgaard, M., Frandsen, F., Pearman, M., Nansen, P., Kold. J., 1999. The

prevalence of gastrointestinal helminths in different poultry production systems. Brit.

Poultry Sci. 40, 439-443.

Permin, A., Ranvig, H., 2001. Genetic resistance to Ascaridia galli infections in chickens.

Vet. Parasitol. 102,101-111.

Petkevičius, S., Knudsen, K.E.B., Murrel, K.D., Wachmann, H., 2003. The effect of inulin

and sugar beet fibre on Oesophagostomum dentatum in pigs. Parasitology. 127, 61-

68.

Petkevičius, S., Knudsen, K.E.B., Nansen, P., Murrel, K.D., 2001. The effect of dietary

carbohydrates with different digestibility on the populations of Oesophagostomum

dentatum in the intestinal tract of pigs. Parasitology. 123, 315-324.

Chapter-I

Background

19

19

Petkevičius, S., Knudsen, K.E.B., Nansen, P., Roepstorff, A., Skjøth, F., Jensen, K., 1997.

The impact of diets varying in carbohydrates resistant to endogenous enzymes and

lignin on populations of Ascaris suum and Oesophagostomum dentatum in pigs.

Parasitology. 114, 555-568.

Ramadan, H.H., Abou Znada, N.Y., 1991. Some pathological and biochemical studies on

experimental Ascaridiasis in chickens. Nahrung-Food. 35, 71-84.

Rehman, H., Hellweg, P., Taras, D., Zentek, J., 2008. Effects of dietary inulin on the

intestinal short chain fatty acids and microbial ecology in broiler chickens as revealed

by denaturing gradient gel electrophoresis. Poultry Sci. 87, 783-789.

Rehman, H.U., Vahjen, W., Awad, W., Zentek, J., 2007. Indigenous bacteria and bacterial

metabolic products in the gastrointestinal tract of broiler chickens. Arch. Anim. Nutr.

61, 319-335.

Riedel, B.B., Ackert, J.E., 1951. Quantity and source of proteins as factors in the resistance

of chickens to Ascarids. Poultry Sci. 30, 497-502.

Roberfroid, M.B., 2005. Introducing inulin-type fructans. Brit. J. Nutr. 93, Suppl. 1, S13-

S25.

Schneeman, B.O., 1999. Fiber, inulin and oligofructose: similarities and differences. J.

Nutr. 129, 1424-1427.

Shakouri, M.D., Kermanshahi, H., Mohsenzadeh, M., 2006. Effect of different non starch

polysaccharides in semi purified diets on performance and intestinal microflora of

young broiler chickens. Int. J. Poultry Sci. 5, 557-561.

Smits, C.H.M., Annison, G., 1996. Non-starch plant polysaccharides in broiler nutrition –

towards a physiological valid approach to their determination. World. Poultry Sci. J.

52, 203-221.

Springer, W.T., Johnson, J., Reid, W.M., 1970. Histomoniasis in gnotobiotic chickens and

turkeys: Biological aspects of the role of bacteria in the etiology. Exp. Parasitol, 28,

383-392.

Stear, M.J., Doligalska, M., Donskow-Schmelter, K., 2007. Alternatives to anthelmintics

for the control of nematodes in livestock. Parasitology. 134, 139-151.

Sundrum, A., Schneider, K., Richter, U., 2005. Possibilities and limitations of protein

supply in organic poultry and pig production. Final Project Report EEC 2092/91

Chapter-I

Background

20

20

(Organic) Revision no. D 4.1 (Part 1), Department of Animal Nutrition and Animal

Health, University of Kassel, Witzenhausen, Germany.

Taylor, M.A., Coop, R.,L., Wall, R.L., 2007. Parasites of poultry and gamebirds p:496. In

Veterinary Parasitology, 3rd Edition. Blackwell Publishing. ISBN: 978-1-4051-1964-

1.

Ten Bruggencate, S. J. M., Bovee-Oudenhoven, I. M. J., Lettink-Wissink, M. L. G.,

Katan, M.B., Van der Meer. R., 2004. Dietary fructo-oligosaccharides and inulin

decrease resistance of rats to salmonella: protective role of calcium. Gut. 53, 530-

535.

Thamsborg, S.M., Roepstorff, A., Larsen, M., 1999. Integrated and biological control of

parasites in organic and conventional production systems. Vet. Parasitol. 84, 169-

186.

Van de Weerd, H.A., Keatinge, R., Roderick, S., 2009. A review of key health-related

welfare issues in organic poultry production. World. Poult. Sci. J. 65, 649-684.

Van der Klis, J.D., Van Voorst, A., Van Cruyningen, C., 1993. Effect of a soluble

polysaccharide (carboxy methyl cellulose) on the physico-chemical conditions in the

gastrointestinal tract of broilers. Br. Poult. Sci. 34, 971-983.

Van Grembergen, G., 1954. Haemoglobin in Heterakis gallinae. Nature. 4418, 35.

Van Krimpen, M.M., Kwakkel, R.P., André, G, Van Der Peet-Schwering, C.M.C., Den

Hartog, L.A., Verstegen, M.W.A., 2007. Effect of nutrient dilution on feed intake,

eating time and performance of hens in early lay. Brit. Poultry Sci. 48, 389-398.

Van Krimpen, M.M., Kwakkel, R.P.,Van der Peet-Schwering, C.M.C., Den Hartog, L.A.,

Verstegen, M.W.A., 2008. Low dietary energy concentration, High nonstarch

polysaccharide concentration, and coarse particle sizes of nonstarch polysaccharides

affect the behavior of feather-pecking-prone laying hens. Poultry Sci. 87, 485-496.


Verstegen, M.W.A., 2009. Effects of nutrient dilution and nonstarch polysaccharide

concentration in rearing and laying diets on eating behavior and feather damage of

rearing and laying hens. Poultry Sci. 88, 759-773.

Chapter-I

Background

21

21

Walker, T.R., Farrell, D.J., 1976. Energy and nitrogen metabolism of diseased chickens:

interaction of Ascaridia galli infestation and vitamin a status. Brit. Poultry Sci. 17,

63-77.

Wallace, D.S., Bairden, K., Duncan, J.L., Fishwick, G., Gill, M., Holmes, P.H., McKellar,

Q.A., Murray, M., Parkins, J.J., Stear, M.J., 1995. Influence of supplementation with

dietary soybean-meal on resistance to Hemonchosis in Hampshire Down lambs. Res.

Vet. Sci. 58, 232-237.

Yegani, M., Korver, D.R., 2008. Factors affecting intestinal health in poultry. Poultry Sci.

87, 2052-2063.

Zentrale Mark- und Preisberichtstelle (ZMP) GmbH, 2008. Marktbilanz, Eier und Geflügel

2008. Bonn, 213 pp.

Chapter-II

Interactions between H. gallinarum and H. meleagridis

22

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CHAPTER - II

Non-starch polysaccharides alter interactions between

Heterakis gallinarum and Histomonas meleagridis

Chapter-II


23

23

Non-starch polysaccharides alter interactions between Heterakis gallinarum and

Histomonas meleagridis

Abstract

Nutrition of the host animal may not only influence interactions between the host and its

parasites, but also relations between different parasites species residing on the same host.

We investigated effects of insoluble and soluble non-starch polysaccharides (NSP) on

establishment and development of Heterakis gallinarum in chicken being treated or left

untreated against Histomonas meleagridis.

Six groups of one-day-old birds were allocated to three diets, two on each diet. The

birds were fed ad libitum either a basal diet (CON), or CON + insoluble NSP (I-NSP) or

CON + soluble NSP (S-NSP) until an age of 11 wk. At an age of 19 d, one of each diet

groups was prophylactically treated for 9 d with dimetridazole (0.05%, w/v) via drinking

water against histomonas. The remaining three groups were left un-treated. Two days after

starting dimetridazole treatment (at 3 wk), each of the 6 groups was divided into two sub-

groups. One dimetridazole treated and one dimetridazole un-treated groups of birds on

each diet (6 groups) were infected with 200 embryonated eggs of H. gallinarum that were

previously harvested from histomonas carrying H. gallinarum infected chickens. The

remaining 6 groups of uninfected birds, either treated or left un-treated against H.

meleagridis, served as controls. Worm burdens of infected birds were determined 8 wk p.i.

Treatment against H. meleagridis significantly increased incidence of H.

gallinarum infection and average worm length in all infected groups independent of the

diet consumed (P<0.001). An interaction between effects of diet and dimetridazole

treatment on worm burden (P<0.001) indicated that the S-NSP diet resulted in lowest

worm burden in dimetridazole un-treated birds, whereas it caused the highest worm burden

in the treated birds (p<0.05). Furthermore, the treatment resulted in higher worm burdens

when compared to un-treated birds on the corresponding diets (p<0.05). Infection with H.

gallinarum impaired body weight (BW) of the chicks (p<0.05) and H. meleagridis

aggravated this effect (p<0.05). Dimetridazole treated and un-treated uninfected birds

developed similar BW (p>0.05). Both NSP supplemented diets resulted in lower (p<0.05)

BW when compared with the CON diet, S-NSP being inferior to I-NSP (p<0.05).

Chapter-II


24

24

It is concluded that H. meleagridis harms the definitive host as well as H.

gallinarum. Both insoluble and soluble NSP supplemented diets favor H. gallinarum

infection while S-NSP additionally intensifies histomonas infection, which then impairs

establishment and development of H. gallinarum.

Keywords: Heterakis gallinarum; Histomonas meleagridis; chicken; vector; host diet;

non-starch polysaccharides.

2.1. Introduction

Because the conventional battery cages will be banned in the European Union by

2012, floor- and outdoor production systems are spreading. However, these systems bear

an increased risk of parasitic infections in poultry (Thamsborg et al., 1999; Fossum et al.,

2009). Heterakis gallinarum, a caecal worm, is one of the most common nematodes in

poultry with a prevalence ranging from 68% to 80% (Permin et al., 1999; Maurer et al.,

2009) especially in organic/free ranging flocks. In spite of its often neglected per se

pathogenicity, the importance of the nematode lies in its role as a main vector for the

transmission of Histomonas meleagridis. The host becomes infected by the ingestion of H.

meleagridis infected embryonated eggs of the nematode (Levine, 1985; McDougald,

2005). Prevalence of histomonas in layer hens in Europe is slightly increasing (Kaufmann-

Bart and Hoop, 2009) and infection outbreaks had not been reported for decades because

layers were kept in cages (Esquenet et al., 2003). Transmission of H. meleagridis among

individuals within or between chicken flocks depends on the presence of H. gallinarum

(McDougald, 2005). In contrast to turkeys a direct transmission of the histomonads, via the

so-called phenomena of cloacal drinking, does not happen in chickens (Hu et al., 2006).

Nutrition of the host animals may not only influence interactions between the host

and its parasites but also relations between different parasites species residing on the same

host. Non-starch polysaccharides (NSP) constitute an important part of dietary fibre.

Dietary NSP are not digested by the endogenous enzymes and, depending on their

fermentability can either less or highly be degraded and utilized by the microorganisms in

the hind parts of the gastrointestinal tract (Englyst, 1989; Schneeman, 1999). Caeca are the

main site of microbial fermentation in chickens (Juskiewicz et al., 2005). Dietary NSP are

known to alter microbial composition in the gastrointestinal tract, particularly in caeca, the

predilection site of H. gallinarum and H. meleagridis. As shown by Petkevičius et al.

Chapter-II


25

25

(1997), NSP varying in intestinal fermentability influence establishment of common

nematode infections of pigs differently. Likewise, an altered caecal environment, induced

by feeding NSP supplemented diets may affect relations between H. gallinarum and H.

meleagridis and their effects on performance in poultry. Therefore we aimed at

investigating the effect of histomonas infection on establishment and development of H.

gallinarum in chickens fed NSP supplemented diets varying in their fermentability. The

objective of the present study was to estimate effects of insoluble and soluble NSP on

interactions between H. gallinarum and H. meleagridis in chicken being treated or not

treated against H. meleagridis.

2.2. Materials and methods

2.2.1. Birds, diets and experimental infections

A total of 360 one-day-old Lohmann Selected Leghorns chicks, obtained from a

commercial hatchery, were used. The one-day-old chicks were weighed together and

randomly divided into 6 feeding groups. The groups of birds were allocated to three diets,

two groups for each diet. The birds were fed ad libitum until an age of 11 weeks (wk)

either a basal diet (CON) supplying recommended metabolizable energy (ME) and

nutrients for grower layers (NRC, 1994) or the basal diet plus insoluble non-starch

polysaccharide (I-NSP) or the basal diet supplemented with soluble non-starch

polysaccharide diet (S-NSP). The I-NSP diet contained additional pea bran meal (Exafine

500, Socode, Belgium) and the S-NSP diet additional chicory root meal (Fibrofos, 60,

Socode, Belgium) as the natural NSP sources. Pea bran and chicory root meal amounted to

9.1% of the I-NSP and S-NSP feed mixtures, respectively. The diets were mixed on air

dry-basis conditions and were pelleted. Each feeding group was kept in a pen scattered

with wood shavings.

At an age of 19 d, a prophylactic treatment with dimetridazole (0.05%, w/v) via ad

libitum offered drinking water was started and continued for 9 d for one group on each

diet. The remaining three groups, each on one diet, were left un-treated. Two days after

starting the dimetridazole treatment, e.g. at an age of 3 wk, each of the 6 groups was

divided into two sub-groups ending up with 12 final experimental groups. One

dimetridazole treated and one dimetridazole un-treated groups of birds on each diet (6

groups) were infected with 200 embryonated eggs of H. gallinarum, which were

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previously harvested from H. gallinarum and concomitant histomonas infected chickens.

The remaining 6 groups of birds, either treated or left un-treated against H. meleagridis,

served as uninfected controls. The experimental structure of the final groups and number of

birds allocated to each group are shown in Table 1.

Table 1. Number of birds allocated to the experimental groups.

Dimetridazole un-treated Dimetridazole treated1

Diet Un-infected Infected2 Un-infected Infected2

CON, N=118 29 30 23 36

I-NSP, N=123 26 34 24 39

S-NSP, N=119 27 30 23 39 1 The birds were treated with dimetridazole (Chevi-col© Pulver, Chevita GmbH, Germany). The compound was given via ad libitum offered drinking water at a concentration of 0.05% (w/v) from 2 d before inoculating H. gallinarum eggs to 7 d post-infection. 2 Each bird was infected with 200 embryonated eggs of H. gallinarum previously harvested from histomonas and concomitant H. gallinarum infected chickens.

2.2.2. Infection material

The infection material was produced at the Department of Animal Science,

University of Goettingen, Germany. Adult female worms, harvested from intestines of

naturally infected chickens collected at different farms, were used as the original material

of infection. The eggs had been used in a previous trial and shown to produce Histomonas-

typical pathological lesions in chickens. Presence of H. meleagridis was macroscopically

and microscopically confirmed in the caecal and liver tissues. The worms were harvested

and used for the present investigation. For embryonation, intact female worms were

incubated at room temperature (20-25 C) for 3 weeks in a media containing 0.5%

(vol/vol) formalin as described by Puellen et al. (2008). After embryonation, the worms

were cut into pieces, and the eggs were squeezed out using a pestle placed on a sieve. The

residual worm tissues on the sieve were flushed and removed, and the eggs were gathered.

The embryonated eggs were stored at + 4°C until the infection day. On the infection day,

the number of eggs/ml aqueous suspension was determined using a McMaster egg

counting chamber. Only eggs in the vermiform and infective larval stages were classed and

counted as embryonated. The counting procedure was repeated five times and the

arithmetic mean was calculated. The infection dose was then adjusted to 200 eggs / 0.2 ml

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of final suspension. Uninfected control birds were given 0.2 ml of 0.5% formalin as

placebo.

2.2.3. Management of the birds

The litter was replaced once (wk 1-3) or twice (wk 4-11) a week. Room

temperature was gradually decreased from 34 C on the first day (d) to 26 C in wk 3 and

thereafter decreased by 2-3C per wk, ending at 18-20 C from wk 6 onwards. A 24 h

lighting period was maintained for the first 2 days and was then reduced to 16 h/d at the

end of the first week. By wk 8, it was reduced to 12h/d and subsequently maintained until

the end of the experiment. At the end of wk 3, the birds were marked with wing tags and

individual body weights (BW) were recorded for the first time and thereafter at weekly

intervals for 5 wk post-infection. Group feed consumption was determined daily. Drinking

water was offered ad libitum. The birds did not get any vaccination or anthelmintic

treatment throughout the experimental weeks. The experimental stable was thoroughly

disinfected 10 d before introducing the birds.

2.2.4. Necropsy

Sentinel birds were subjected to necropsies to determine infection induced macro-

and microscopical lesions and to detect H. meleagridis in caecal sections. For this purpose,

4-5 birds from each of 12 experimental groups were examined in wk 2, 3, and 5 p.i.,

respectively. The caecal samples were fixed for 24 hours in 4% phosphate-buffered

formalin, embedded in paraffin and processed for Hematoxylin and Eosin (H&E) staining

according to standard methods. Tissue sections were examined microscopically for

infection induced lesions such as epithelial erosion and ulceration, lymphocyte and

heterophil infiltrations as well as for the presence of the histomonads in the tissue. The

sentinel birds are not included in the animal numbers shown in Table 1.

2.2.5. Worm harvest

All the birds were slaughtered after electrical stunning 8 wk p.i. After slaughtering,

the gastrointestinal tract was removed, caeca were separated and the worm burdens were

quantified by the procedure described by Gauly et al. (2008). Briefly, caeca were opened,

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the content was removed, and the caecal walls were flushed to remove the worms. The

caecal content was flushed with low-pressure tap water through a sieve with a mesh

aperture of 100 µm, and the residues were transferred into Petri dishes to be examined by

using a stereomicroscope. Average intact worm length was estimated by measuring 10

female and 10 male randomly selected worms per each bird. In cases of lower than 10

worms per sex were available, all the intact worms were measured. Caeca from uninfected

control birds (20% of each group) were also processed to confirm infection free status of

the controls.

2.2.6. Chemical analyses of the diets

The composition, nutrient and energy contents of the experimental diets are given

in Table 2. At weeks 1, 6 and 11, representative feed samples were taken and analyzed for

dry matter (DM), crude ash (CA), crude protein (CP), sugar, starch, and ether extract (EE)

using standard methods (Naumann and Bassler, 1997). Neutral and acid detergent fibre

(NDF and ADF, respectively) were analyzed according to Van Soest et al. (1991) and

results are given exclusive of ash. The metabolizable energy of the diets (MJ ME/kg DM)

was calculated (FMVO, 2008). Insoluble and soluble non-starch polysaccharides were

measured using an enzymatic test (Megazyme, 2007). Inulin was determined according to

Naumann and Bassler (1997).

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Table 2. Composition and analysis of the experimental diets.

Item CON1 I-NSP2 S-NSP3 Components, g/kg (as fed-basis)

Barley 290 264 264 Wheat 540 491 491 Fishmeal4 80 73 73 Casein 45 41 41 Soybean oil 20 18 18 Premix5 10 9 9 MCP 9 8 8 CaCO3 6 5 5 Pea bran6 - 91 - Chicory root meal7 - - 91

Analyzed composition DM, g/kg 896 901 894

Nutrient, g/kg DM Ash 54 52 54 CP 222 207 207 NDF 115 164 110 ADF 31 86 41 Ether extract 38 35 36 Starch 491 439 411 Insoluble NSP 102 170 104 Soluble NSP 18 22 24 Inulin - - 70

ME, MJ/kg DM8 13.28 12.05 11.85

1 Basal diet. 2 Insoluble non-starch polysaccharide supplemented diet = 1000 g CON plus 100 g pea bran. 3 Soluble non-starch polysaccharide supplemented diet = 1000 g CON plus 100 g chicory root meal. 4 Fishmeal; 64% CP and 8% CL. 5 Supplied per kg of premix: 1.200.000 IU vitamin A, 350.000 IU vitamin D3, 4.000 mg vitamin B1, 800 mg

vitamin B2, 600 mg vitamin B6, 3.200 mg vitamin B12, 450 mg vitamin K3, 4.500 mg nicotinic acid, 1.500 mg Ca-pantothenate, 120 mg folic acid, 5.000 mg biotin, 55.000 mg choline chloride, 3.200 mg Fe, 3.200 mg Fe-(II)-Sulphate, 1.200 mg Cu-(II)-sulfate pentahydrate, 10.000 mg Mn-(II)-oxide, 8.000 mg Zn-Oxide, 160 mg iodine, 160 mg Ca-iodine-hexahydrate, 40 mg Na-Selenite, 64 mg Cobalt, 64 mg basic Co-(II)-Carbonate-monohydrate, 10.000 mg BHT (Product code: 77046, Vilomix, Germany).

6 Pea bran: contained 86.9% crude fibre (Exafine 500, Socode, Belgium). 7 Chicory root meal: average polymerization degree (DP) of inulin = 9. (Fibrofos 60, Socode, Belgium). 8 ME = metabolizable energy, MJ/kg DM= [( g CP/kg DM x 0.01551) + (g CL/kg DM x 0.03431) + (g

starch/kg DM x 0.01669) + (g sugar/kg DM x 0.01301)]. Sugar contents of the diets were estimated based on sugar contents of the components.

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2.2.7. Statistics

2.2.7.1. Incidence of H. gallinarum infection

Effects of the diets on the incidence of H. gallinarum infection (proportion of

worm-harboring birds to the experimentally infected birds) were analyzed using

GENMOD procedure of SAS (2010) with a logit link function. The GENMOD procedure

fits the generalised linear models and suited for responses with binary outcomes (Kaps and

Lamberson, 2004). Because all the dimetridazole treated birds of I-NSP and S-NSP fed

birds harbored worm(s), the infected groups were not comparable for the incidence of

infection within dimetridazole treated groups. Therefore effect of diet on the incidence of

infection was estimated within dimetridazole untreated groups. To find out the effect of

dimetridazole treatment on the incidence of H. gallinarum infection, each dimetridazole

treated infected group was compared with its un-treated corresponding group on the same

diet.

2.2.7.2. H. gallinarum worm burden and worm length

Transformation with a natural logarithm (ln) function [ln(y) = ln(y+1)] was applied

to worm burden data that were positively skewed (Skewness > 0) and showed non-normal

distribution (Kolmogorow-Smirnow, p<0.05) to correct for heterogeneity of variance and

to produce approximately normally distributed data. After transformation the variances

were still not equal among the groups. Therefore the transformed data were analyzed with

a mixed model (Proc Mixed), by which unequal variances were taken into account. This

approach improved fit statistics of the model as indicated with smaller BIC and AIC

values. The statistical model for the worm burden, worm length and sex ratio included

fixed effects of diet (1-3), Dimetridazole treatment (0, 1), interaction effect between diet

and dimetridazole treatment, and the residual error term.

2.2.7.3. Growth and feed utilization data

Body weight (BW) and feed:gain ratio of the birds were evaluated for a period of

five weeks (p.i.), covering the pre-patent period of the nematode. The data were analyzed

with a 3-way ANOVA that included fixed effects of diets (CON, I-NSP, S-NSP), H.

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gallinarum infection (infected, not infected), the dimetridazole treatment (untreated,

treated), all possible interactions between these factors and the residual error term. Because

effects of infection(s) and dimetridazole treatment could only be estimated for the post-

infectional period, BW and feed:gain of the birds in the pre-infectional period was

analyzed with another model. This model included fixed effect of diet and block effect of

double feeding system for each diet (two separate groups were on each diet) in this period.

2.2.7.4. Presentation of the results and multiple comparison tests

After infection at the end of wk 3, the birds were kept according to a 3 x 2 x 2

factorial arrangement of treatments with diet, infection and the dimetridazole treatment as

the main factors. Therefore, unless no significant interactions between the effects of the

main experimental factors were encountered, the data are presented as the main effects of

diet, infection and the dimetridazole treatment. In case of significant interactions between

the main factors were encountered, the results are presented for the corresponding single

groups under influence of the interacting factors.

Tukey-Kramer test was used to partition differences among feeding groups when a

significant non-interactive main effect was encountered. In cases of significant

interactions, sub-groups were separated using the Tukey-Kramer post-hoc test

(alpha=0.05). All the statistical analyses were performed with SAS (2010).

2.2.8. Ethical consideration

The experimental procedures followed the animal welfare rules. The infection dose

(200 eggs) given to each bird was within the range of the worm burdens that can be

observed in natural sub-clinical infections. The procedures for experimental infections

followed the guidelines suggested by the World Association for the Advancement of

Veterinary Parasitology for evaluating the effectiveness of anthelmintics in chickens and

turkeys (Yazwinski et al., 2003).

2.3. Results

2.3.1. Incidence of H. gallinarum infection

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As shown in Figure 1, dimetridazole treatment increased incidence of H.

gallinarum infection in all the feeding groups (P<0.001). Without dimetridazole treatment,

there was an effect of diet on the incidence of infection (P=0.0014). The I-NSP diet led to

higher incidence of H. gallinarum infection in comparison to S-NSP (p<0.05). Further, the

incidence of infection in the CON group tended to be lower than in the I-NSP group

(p=0.059) and higher than in the S-NSP group (p=0.089). Dimetridazole treatment resulted

in a high incidence of H. gallinarum infection (>90%) in all three feeding groups with

insufficient variation for comparisons within dimetridazole treated groups.

2.3.2. H. gallinarum establishment rate

Establishment rates of the H. gallinarum eggs were lower than 7% in all the dimetridazole

un-treated feeding groups, whereas dimetridazole treatment resulted in 12.1%, 17.7% and

27.4% establishment rates after feeding CON, I-NSP and S-NSP, respectively (Figure 2).

y

(ab)-x

y

b-x

y

a-x

0

20

40

60

80

100

Dimetridazole (-) Dimetridazole (+)

Inc

ide

nc

e,

%

CON

I-NSP

S-NSP

Figure 1. Incidence of H. gallinarum infection without (-) and with (+) dimetridazole

treatment of the birds on different diets (n= 30-35 per group).

ab : Dimetridazole un-treated groups sharing no common letters differ (p<0.05). (ab) : Groups sharing the same letters in brackets tend to differ (a: p=0.059; b: p=0.089). xy : Dimetridazole treated or un-treated groups on the same diet with no common letter

differ (P<0.001).

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0

10

20

30

40

Dimetridazole (-) Dimetridazole (+)

Est

ablis

hm

ent

rate

, %

CON I-NSP S-NSP

Figure 2. Establishment rate (%) of Heterakis gallinarum after a single dose (200

eggs/bird) inoculation of Histomonas meleagridis positive eggs in chickens, left untreated

(-) or treated (+) with dimetridazole (n= 30-35 per group, means and standard deviations

on the error bars).

2.3.3. Average worm burden

Uninfected control birds were free of H. gallinarum as confirmed by examination

of the cecal contents. As shown in Table 3, there was a significant interaction between diet

and the dimetridazole treatment on worm burdens of the birds (P<0.001). Dimetridazole

treated S-NSP fed birds harboured higher number of worms than all dimetridazole treated

and un-treated groups of birds (p<0.05). All the dimetridazole treated groups had higher

worm burdens in comparison to the corresponding un-treated feeding groups (p<0.05).

Without dimetridazole treatment, I-NSP-fed birds had higher worm burdens than S-NSP-

fed birds (p<0.05). With dimetridazole treatment, S-NSP led to higher worm burdens than

CON and I-NSP (p<0.05). Dimetridazole treated I-NSP-fed birds tended to harbour higher

numbers of worms than the CON-fed birds (p=0.093). Worm burdens of dimetridazole un-

treated I-NSP fed birds and dimetridazole treated CON fed birds did not differ (p>0.05),

whereas dimetridazole treated I-NSP fed birds had higher worm burdens than all the

dimetridazole un-treated groups (p<0.05). Without dimetridazole treatment, no larval

stages were found in the S-NSP-fed birds and only a few in the CON and I-NSP fed birds

(3-4 birds). Dimetridazole application resulted in 0.1 larvae per bird after feeding CON,

whereas I-NSP and S-NSP fed birds harboured 3.5 and 2.1 larvae, respectively.

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2.3.4. Sex ratio and worm length

Proportion of numbers of female to male worms (sex ratio) was, in general, in favor

of the male worms (Table 4). No significant effects of diet, dimetridazole treatment or

interaction between these two factors on the sex ratio were observed (P>0.05). Male and

female worm length remained unaffected by the type of diet (P>0.05), however,

dimetridazole treatment led to an increase in length of the male and female worms

irrespective of the type of diet consumed by the birds (P<0.001). There was no interaction

effect of diet and dimetridazole treatment on worm length (P>0.05).

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Table 3. Interaction of the diets and dimetridazole treatment on average H. gallinarum worm burdens in dimetridazole treated and untreated birds1.

Without dimetridazole With dimetridazole P-values, ≤

Log-worm burden CON I-NSP S-NSP CON I-NSP S-NSP Diet Dimetridazole Interaction

LS-MEANS 0.94ab 1.62bc 0.45a 2.50cd* 3.28d* 3.85e SE 0.236 0.240 0.144 0.254 0.156 0.115

0.006 0.001 0.001

1: n = 30-35 per each infected group. (abcde): Groups with no common superscript differ (Tukey, p<0.05). (*): Groups sharing the sign tend to differ (Tukey, p = 0.093).

Table 4. Effects of the diets and the dimetridazole treatment on sex ratio and average worm length.

Diets1 Dimetridazole treatment2

Item CON I-NSP S-NSP PSE3 P, ≤ (-) (+) PSE2 P, ≤

Interaction P, ≤

Sex ratio (N=133) 0.84 0.95 0.87 0.134 0.750 0.87 0.90 0.121 0.773 0.818 Average male length, mm (N=117) 9.12 9.38 8.96 0.379 0.368 8.45a 9.86b 0.282 0.001 0.4916 Average female length, mm (N=115) 10.60 10.81 10.29 0.280 0.111 9.44a 11.70b 0.224 0.001 0.2244

(ab): Groups with no common superscript in a row within a factor differ (Tukey, p<0.05). 1 CON = basal diet; I-NSP = 1,000 g CON + 100 g pea bran; S-NSP = 1,000 g CON + 100 g chicory root meal. 2 The birds were treated with dimetridazole (Chevi-col© Pulver, Chevita GmbH, Germany). The compound was given via ad libitum offered drinking water at a concentration of

0.05% (w/v) from 2 d before inoculating H. gallinarum eggs to 7 d post-infection. 3 Pooled SE.

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2.3.5. Mortality, clinical observations and pathology

The overall mortality (up to wk 11) was less than 3% and most of the deaths

occurred in the first week of the pre-infectional period (2.5%). Following the experimental

infection, there was a decrease in feed intake of the infected birds that were left un-treated

against histomonas, whereas only a slight decrease was observed in association with

dimetridazole application (Figure 3).

Due to the limited number of sentinel birds at each of the examination weeks, the

intensity of pathological and histo-pathological lesions at the caecal level could not be

quantified for each feeding group. Dimetridazol untreated infected birds showed

macroscopic lesions such as thickening of the caecal wall and fibrinous to fibrino-

hemorrhagic exudates in the caecal lumen. Histologically, severe hyperplasy of tunica

muscularis, numerous histomonads, massive lymphocyte, heterophil and macrophage

infiltrations, as well as coagulation necrosis have been observed in 2 and 3 wk p.i.. At 5 wk

p.i., the severity of the lesions declined. After dimetridazole treatment, macroscopical

lesions were not observed in either of the feeding groups. Microscopicaly, dimetridazole

treated infected birds from all feeding groups exhibited mild to moderate lesions of the

caecal tissue i.e., lymphocytes infiltration in the lamina propria and formation of lymphoid

aggregations. Histomonads were absent. Uninfected birds, either left untreated or treated

with dimetridazole, were negative for histomonads and the pathological lesions.

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Figure 3. Average daily feed intake of H. gallinarum infected (+) and uninfected control

(-) groups on different diets, without and with the dimetridazole treatment.

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2.3.6. Host animal growth performance and feed utilization in the pre-infectional period

During the pre-infection period (1-3 wk), birds being supplied with I-NSP

consumed 2.8 % more feed compared to CON (Table 5). No significant effect of double

feeding system for each diet was found for BW and feed:gain ratio in the pre-infectional

period (P>0.05). CON-fed birds had higher body weights than S-NSP fed birds (p<0.05),

whereas I-NSP fed birds did not differ from birds of CON- or S-NSP-groups (p>0.05).

Feed:gain ratio was smaller in CON than in NSP fed birds (p<0.05).

Table 5. Effects of diet on feed intake, body weight (BW), and feed:gain ratio in the pre-

infectional period (1-3 wk).

Diet1 Item CON I-NSP S-NSP PSE 2 P-value

Feed consumption3, g/bird 351 361 351 - - BW4, g 212a 207ab 203b 1.533 0.001 Feed:gain, g/g 2.03a 2.14b 2.14b 0.020 0.001

1 CON = basal diet; I-NSP = 1,000 g CON + 100 g pea bran; S-NSP = 1,000 g CON + 100 g chicory root meal. 2 Pooled SE. 3 Calculated from daily group consumptions. 4 Body weight at the end of wk 3 of life. The average one-day-old weight was 37 g.

2.3.7. Host animal growth performance and feed utilization up to 5 wk p.i.

As shown in Table 6, the effects of diet on BW and feed:gain were significant

(P<0.001). Feeding CON resulted in higher BW and lower feed:gain ratios in comparison

to feeding the NSP diets. Feeding I-NSP resulted in higher BW development but also

higher feed:gain ratio in comparison to feeding S-NSP (p<0.05). There were significant

interactions between effects of dimetridazole treatment and infection for BW and feed gain

ratio (P<0.05). Uninfected birds with and without dimetridazole treatment had almost the

same BW (p>0.05) whereas infected birds were lighter (p<0.05). Dimetridazole un-treated

infected birds had lower BW than the dimetridazole treated infected birds (p<0.05). The

application of dimetridazole decreased the feed:gain ratio of infected and uninfected birds

compared to the un-treated birds (p<0.05). Infected un-treated birds had higher feed:gain

ratio than the uninfected un-treated birds (p<0.05).

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Table 6. Effects of the investigated factors on feed consumption, body weight (BW), and

feed:gain ratio (as LSMEANS and SE).

Feed intake1 BW2 Feed:gain Item g/bird g/bird SE g/g SE Diet

CON 1775 706a 4.851 2.67a 0.023 I-NSP 1886 683b 4.783 2.95b 0.023 S-NSP 1793 665c 4.856 2.88c 0.023

Infection3 x dimetridazole4 Un-infected / dimetridazole (-) 1931 708a 5.752 2.86a 0.028 Infected / dimetridazole (-) 1778 633c 5.377 2.98b 0.026 Un-infected / dimetridazole (+) 1827 709a 6.221 2.74c 0.030 Infected / dimetridazole (+) 1767 688b 4.877 2.74c 0.023

P-values5 Diet - 0.001 0.001 Infection - 0.001 0.038 Dimetridazole treatment - 0.001 0.001 Diet x infection - 0.563 0.687 Diet x dimetridazole - 0.950 0.721 Infection x dimetridazole - 0.001 0.019 Diet x infection x

dimetridazole - 0.951 0.412

(abc): Values with no common superscripts within a factor indicate differences (Tukey, p<0.05). 1 Calculated from daily group consumptions. 2 Body weight at the end of wk 8 of life, i.e., at 5 wk p.i. . 3 The birds were treated with dimetridazole (Chevi-col© Pulver, Chevita GmbH, Germany). The compound was given via ad libitum offered drinking water at a concentration of 0.05% (w/v) from 2 d before inoculating H. gallinarum eggs to 7 d post-infection. 4 H. gallinarum infection with 200 embryonated eggs of H. gallinarum previously harvested from histomonas and concomitant H. gallinarum infected chickens. 5 P-values derived from the 3-way ANOVA analysis. LSMEANS and SE are presented either for significant non-interactive main effects (Diet) or for significant interactions between any of the main factors (in this case, interaction between infection x dimetridazole treatment).

2.4. Discussion

Under natural conditions, the vast majority of Heterakis eggs from chicken are H.

meleagridis positive (McDougald, 2005). The minimum possible infection rate, i.e., the

proportion of Histomonas-carrying H. gallinarum eggs that are obtained from sub-

clinically infected birds and those are able to produce clinical histomonosis, has been

estimated to be 1:139 (Lund, 1958). In any case, it can be assumed that the histomonas

proven positive infection dose used led to a double infection in all infected birds. This is

confirmed by the presence of the protozoon and histomonas-associated lesions on caecal

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tissues of the sentinel birds examined by necropsy. Dimetridazole is known to be highly

effective against H. meleagridis in chicken (Hu and McDougald, 2004). Histomonads are

released from the larva during molting rather than at death of larvae (McDougald, 2005).

Lund (1968) showed that the histomonads are liberated from the larva starting as early as

4.5 d after inoculation of the eggs. Therefore, the dimetridazole treatment in the present

study starting from 2 d before infection to 7 d p.i. can be regarded as effective to eliminate

histomonas infection in the treated birds.

The results of the present study indicate that H. meleagridis decreased incidence,

establishment rate and development of H. gallinarum in all feeding groups when compared

with the corresponding histomonas free feeding groups. This is in agreement with

observations of Lund (1958) who reported that caeca with clinical evidence of blackhead

disease (histomonosis) contain fewer or no worms at all when compared to unaffected

caeca in chickens and turkeys. We measured a decrease by almost 20% in length of

histomonas infected heterakis females. Lund (1958) reported a decline by 5.1% in worm

length of histomonas infected female H. gallinarum. Female worm length is the best

predictor of fecundity in H. gallinarum (Tompkins and Hudson, 1999). Though

quantification of female worm fecundity was not in the scope of this study, histomonas

infection might have impaired female worm fecundity as well. Detrimental effects of

parasites on their vectors are known for a wide range of parasite-vector systems (Elliot et

al., 2003). For instance, malaria-infected mosquitoes are less fit as to longevity and

reproductive success than unaffected ones (Hogg and Hurd, 1997; Hurd et al., 2005).

Although the evolutionary mechanisms behind detrimental effects of parasites on their

vectors are not fully understood, it might be hypothesized that H. meleagridis exerts a

selection pressure on H. gallinarum resulting in a more suitable worm population for the

transmission of histomonads.

The type of diet played an important role in the interaction between the two parasites,

since feeding S-NSP resulted in lower incidence and total worm burden than I-NSP in case

of dual infection, but this diet resulted in highest worm burden when histomonas was

eliminated. The effect of S-NSP high probably indicates the involvement of the caecal

bacteria in the relations of the two parasites. Bacteria play critical roles in the life cycle of

the two parasites (McDougald, 2005). H. meleagridis attains full virulence in the caeca of

chicken only in combination with the presence of several bacteria species (Springer et al.,

1970; McDougald, 2005). Springer et al. (1970) showed that Heterakis larvae were not

even able to survive when injected into caeca of gnotobiotic birds, indicating that the role

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of bacteria is more vital for the survival of Heterakis than of histomonads. Moreover, H.

gallinarum is considered as a bacteria feeder (Bilgrami and Gaugler, 2004). The S-NSP

diet contained inulin, a highly fermentable substance which acts as a prebiotic and has

been shown to increase intestinal bacteria counts and their metabolic activity in turkeys

(Juskiewicz et al., 2005) and in chickens (Rehman et al., 2008a). Fermentation of inulin by

the microorganisms markedly changes caecal environment in terms of a lowered pH and

altered profile of short chain fatty acids that consequently increase caecal size in chicken

(Juskiewicz et al., 2005; Rehman et al., 2008a, b). It can be assumed that abundance of

caecal bacteria induced by feeding S-NSP may have encouraged the development of

histomonas infection. Lower incidence and worm burdens as well as retarded worm growth

in histomonas infected birds may be explained by the heavy pathological lesions which can

be considered as a harsh environment for H. gallinarum to establish and to further develop.

On the other hand, S-NSP favored the establishment of H. gallinarum in the absence of

histomonads as indicated by the highest worm burden in the birds treated against H.

meleagridis.

Both, dual and single infections resulted in lower body weight development of the

birds. This might have been caused by partial diversion of nutrients from growth to

development of immunity (Kyriazakis and Houdijk, 2006), and to support repair of

damaged mucosal structures of the intestine in infected birds (Hoste, 2001). Moreover, the

infection induced a decrease in feed intake particularly in the dimetridazole un-treated

birds, being associated with a reduced intake of ME and essential nutrients such as

essential amino acids, minerals, trace elements and vitamins, may have contributed to the

retarded body weight development of the birds in all feeding groups.

Although a single H. gallinarum infection impaired body weight development of the

birds, H. meleagridis aggravated this effect. Though in dimetridazole un-treated birds the

S-NSP diet resulted in lower infection rate and worm burden than I-SNP, I-NSP led to

higher body weight development than S-NSP. This may also suggest that S-NSP

intensified histomonas infection which then impaired establishment and development of H.

gallinarum, exerting an additional negative effect on host growth performance.

2.5. Conclusion

Histomonas meleagridis harmed the definitive host as well as H. gallinarum.

Insoluble and soluble NSP supplemented diets favored H. gallinarum infection while S-

Chapter-II


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42

NSP additionally intensified histomonas infection, which then impaired establishment and

development of H. gallinarum. Therefore, dietary NSP appear not to be suited to protect

chicken against infections with H. gallinarum and H. meleagridis.

References



Elliot, S.L., Adler, F.R., Sabelis, M. W., 2003. How virulent should a parasite be to its

vector? Ecology, 84, 2568-2574.



Esquenet, C., De Herdt, P., Bosschere, H., Ronsmans, S., Ducatelle, R., Van Erum, J.,

2003. An outbreak of histomoniasis in free-range layer hens. Avian Pathol. 32, 305-

308.



doi:10.1186/1751-0147-51-3

Futtermittelverordnung (FMVO), 2008. Anlage 4. Shätzgleichungen zur Berechnung des

Energiegehaltes von Mischfuttermitteln. URL: http://www.gesetze-im-

internet.de/futtmv_1981/anlage_4_76.html (Accessed on 15.07.2010).

Gauly, M., Kanan, A., Brandt, H., Weigend, S., Moors, E., Erhardt, G., 2008. Genetic

resistance to Heterakis gallinarum in two chicken layer lines following a single dose

of infection. Vet. Parasitol. 155, 74-79.

Hogg, J.C., Hurd, H., 1997. The effects of natural Plasmodium falciparum infection on the

fecundity and mortality of Anopheles gambiae s. l. in north east Tanzania.




Hu, J., Fuller, L., Armstrong, P.L., McDougald, L.R., 2006. Histomonas meleagridis in

chickens: attempted transmission in the absence of vectors. Avian Dis. 50, 277-279.

http://www.gesetze-im-internet.de/futtmv_1981/anlage_4_76.html�


Chapter-II


43

43

Hu, J., McDougald, L.R., 2004. The efficacy of some drugs with known antiprotozoal

activity against Histomonas meleagridis in chickens. Vet Parasitol, 121, 233-238.

Hurd, H., Taylor, P.J., Adams, D., Underhill, A., Eggleston, P., 2005. Evaluating the costs

of mosquito resistance to malaria parasites. Evolution. 59, 2560-2572.



inulin. Arch. Gefluegelkd. 69, 175-180.

Kaps, M., Lamberson, W. R., 2004. Biostatistics for Animal Science, pp. 394-412. CABI

Publishing, Wallingford, U.K., 445 pp.

Kaufmann-Bart, M., Hoop, R.K., 2009. Diseases in chicks and laying hens during the first

12 years after battery cages were banned in Switzerland. Vet. Rec. 164, 203-207.



Levine, 1985. Veterinary Protozoology, Chapter 4, pp. 86. First Edition. The Iowa State

University Press, Ames, Iowa, 414 pp.

Lund, E.E., 1958. Growth and development of Heterakis gallinae in turkeys and chickens

infected with Histomonas meleagridis. J. Parasitol. 44, 297-301.

Lund, E.E., 1968. Acquisition and liberation of Histomonas wenrichi by Heterakis

gallinarum. Exp. Parasitol. 22, 62-67.

Maurer, V., Amsler, Z., Perler, E., Heckendorn, F., 2009. Poultry litter as a source of

gastrointestinal helminth infections. Vet. Parasitol. 161, 255-260.


Avian Dis. 49, 462-476.

Megazyme, 2007. Total dietary fibre assay procedure. Megazyme International Ireland

Ltd., Wicklow, Ireland.

National Research Council (NRC), 1994. Nutrient requirements of poultry. National

Academy Press, Washington, D.C, 157 pp.

Naumann, K., Bassler, R., 1997. Methodenbuch. Die chemische Untersuchung von

Futtermitteln. Band III. VDLUFA-Verlag, Darmstadt, Deutschland.

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44








Puellen, U., Chat, S., Moors, E., Gauly, M., 2008. The role of preparation technique,

culture media and incubation time for embryonation of Heterakis gallinarum eggs.

Deut. Tierarztl. Woch. 115, 30-33.

Rehman, H., Böhm, J., Zentek, J., 2008a. Effects of differentially fermentable

carbohydrates on the microbial fermentation profile of the gastrointestinal tract. J.

Anim. Physiol. Anim. Nutr. 92, 471-480.

Rehman, H., Hellweg, P., Taras, D., Zentek, J., 2008b. Effects of dietary inulin on the



SAS Institute Inc., 2010. SAS OnlineDoc® Version 9.1.3, Cary, NC, USA.


Nutr. 129, 1424-1427.



383-392.



186.

Thompkins, D.M., Hudson, P.J., 1999. Regulation of nematode fecundity in the ring-

necked pheasant (Phasianus colchicus): not just density dependence. Parasitology.

118, 417-423.

Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral

detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy

Sci. 74, 3583-3597.

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45

Yazwinski, T.A., Chapman, H.D., Davis, R.B., Letonja, T., Pote, L., Maes, L., Vercruysse,

J., Jacobs, D.E., 2003. World Association for the Advancement of Veterinary

Parasitology (W.A.A.V.P.) guidelines for evaluating the effectiveness of

anthelmintics in chickens and turkeys. Vet. Parasitol. 116, 159-173.

Chapter-III

Effects of NSP on H. gallinarum

46

46

CHAPTER - III

Effects of dietary non-starch polysaccharides on establishment and fecundity of

Heterakis gallinarum in grower layers

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47

Effects of dietary non-starch polysaccharides on establishment and fecundity of

Heterakis gallinarum in grower layers

Abstract

It was hypothesized that the establishment and fecundity of Heterakis gallinarum in

chicken may be affected by dietary non-starch polysaccharides (NSP), which are known to

alter the intracaecal environment. Therefore, a total of 670 one-day-old female layer chicks

were fed ad libitum for 11 wk one of the following experimental diets. The birds were fed

either a basal diet (CON) or a basal diet plus pea bran rich in insoluble NSP (I-NSP), or a

basal diet plus chicry root meal as a source of inulin rich soluble NSP (S-NSP) in a three-

times repeated experiment. At the end of wk three, each feeding group was subdivided into

an uninfected and an infected group of birds each inoculated with 200 embryonated eggs of

H. gallinarum. All the birds were slaughtered 8 wk post infection, and their worm burdens,

as well as the nematode egg excretion were determined.

The NSP supplemented diets and also infection led to reduced body weights (BW)

of birds and impaired the feed conversion rate (P<0.001). The NSP supplemented diets

increased average length of caecum (P<0.001), with S-NSP exerting a stronger effect than

I-NSP (P<0.05). Full caeca weight was increased by S-NSP (P<0.001). The infection

increased the weight of full and empty (washed) caeca (P≤0.027). Feeding S-NSP lowered

intracaecal pH and molar proportion of acetate and increased that of butyrate compared to

CON and I-NSP (P<0.001). Caecal pool of volatile fatty acids (VFA) was increased with

S-NSP (P<0.001). Infection increased intracaecal pH (P=0.002) accompanied by lower

molar proportion of butyrate (P<0.001) and reduced caecal pools of VFA (P<0.001).

The NSP-diets elevated incidence of infection (P<0.01), average number of larvae

(P<0.009) and total worm burden (P<0.001) compared to CON. The worm length was not

influenced by the diet (P>0.05). The daily amount of faeces increased in NSP-fed birds

(P<0.001). Number of eggs per gram of faeces (EPG), number of eggs excreted per worm

population of a bird within 24 h (EPD) and female worm fecundity (EPD/female worm)

were elevated after feeding S-NSP (P≤0.002), whereas I-NSP led to lower EPG/female

worm (P<0.05). The EPD increased in the sequence of CON < I-NSP < S-NSP (P<0.001).

It is concluded that the pea bran and chicory root meal used as sources of insoluble

and soluble dietary NSP, respectively, provided favorable conditions for the establishment

Chapter-III


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48

of H. gallinarum in grower layers. Chicory root meal additionally enhanced fecundity of

the nematode. Therefore, the two natural sources of insoluble and soluble NSP offer no

potential as protecting agents against H. gallinarum infections in chicken.

Keywords: Non-starch polysaccharides; Heterakis gallinarum; worm fecundity; pea bran;

chicory root; inulin; chicken; caeca.

3.1. Introduction

The diet can alter the interactions between the host animals and their parasites

(Coop and Holmes, 1996; Coop and Kyriazakis, 1999; Stear et al., 2007). Dietary vitamins

(Idi et al., 2007), minerals (Gabrashanska et al, 2004), protein or amino acids (Riedel and

Ackert, 1951; Daş et al., 2010) have been shown to influence infections of poultry with the

common fowl parasite Ascaridia galli. Dietary non-starch polysaccharides (NSP)

influenced infections of pigs with Oesophagostomum dentatum (Petkevičious et al., 1997;

2001; 2003) and of chickens with Ascaridia galli (Daenicke et al., 2009). As NSP are only

degradable by the intestinal microbiota (Englyst, 1989), their effects on nematode

infections should mainly be ascribable to alterations of digesta characteristics and intestinal

microbial fermentation.

In recent years, Heterakis gallinarum has become more important with the

increasing number of poultry kept in floor husbandry systems, where the prevalence of this

parasite may reach 80% (Permin et al., 1999; Maurer et al., 2009). The nematode is known

as the main vector for the transmission of Histomonas meleagridis, which is brought about

by the ingestion of embryonated eggs of the nematode by the host animal (McDougald,

2005). Dietary NSP have been shown to interfere with the interrelation between H.

gallinarum and Histomonas meleagridis (Daş et al., 2009). Therefore, it must be ensured

that the nematode is free of H. meleagridis if dietary effects on infection parameters for the

worm are investigated.

The caeca are the main sites of microbial fermentation in poultry (Józefiak et al.,

2004) and are the predilection sites of H. gallinarum. We hypothesized that the

establishment and fecundity of H. gallinarum may be affected and regulated by dietary

NSP, which are known to alter the intracaecal environment. Therefore, the objective of the

present investigation was to examine the effects of low or highly fermentable NSP on the

establishment and fecundity of the nematode as well as on parameters of caecal

Chapter-III


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fermentation and performance of grower layers experimentally infected with H.

gallinarum.

2. Material and methods

3.2.1. Experimental design, diets and management of the birds

In a three times repeated experiment, conducted in the years 2008 - 2009, a total of

670 one-day-old female Lohmann Selected Leghorn (LSL) chickens were used. The chicks

were weighed together and divided within each repetition into 3 feeding groups. Each

feeding group was fed ad libitum one of the following pelleted experimental diets (Table

1): basal diet (CON), basal diet plus insoluble NSP (I-NSP), and basal diet plus soluble

NSP (S-NSP) from hatch until wk 11 of life. Insoluble NSP were supplied by mixing on air

dry-basis one kg CON with 100 g pea bran (Exafine 500, Socode, Belgium). For S-NSP,

one kg CON was mixed with 100 g chicory root meal (Fibrofos 60, Socode, Belgium).

Daily feed consumption was monitored per group. Drinking water was offered ad libitum.

Until wk three, each feeding group was kept in a pen scattered with wood shavings.

The litter was replaced once (wk 1-3) or twice (wk 4-11) a week. Room temperature was

gradually decreased from 34 C on the first day (d) to 26 C in wk three and thereafter

decreased by 2-3C per wk, ending at 18-20 C from wk six onwards. A 24 h lighting

period was maintained for the first two days and was then reduced to 16 h/d at the end of

the first week. By wk eight, it was reduced to 12h/d and subsequently maintained until the

end of the experiment. At the end of wk three, the birds were marked with wing tags and

individual body weights (BW) were determined for the first time and thereafter at weekly

intervals.

3.2.2. Experimental infection

The inocula were prepared at the Department of Animal Science, University of

Goettingen, Germany. Adult female worms, harvested from intestines of naturally infected

chickens from different farms, were used as the original source of infection material. The

infection material was passed for one generation in a preliminary animal trial in which a

dimetridazole treatment was applied to chickens to eliminate possible contamination of

Histomonas meleagridis. Controls confirmed that the new batch of infection material was

obtained from H. meleagridis free Heterakis-infected birds. For embryonation, the second

generation female worms were incubated at room temperature (20-25 C) for three weeks

in media containing 0.1% (wt/vol) potassium dichromate (K2Cr2O7) as described by

Puellen et al. (2008). After embryonation, the worms were cut into pieces, and the eggs

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50

were squeezed out using a pestle placed on a sieve. The residual worm tissues on the sieve

were flushed away, and the eggs were collected. The embryonated eggs were stored at 4 C

until the infection day. On the infection day, the number of eggs/ml aqueous suspension

was determined using a McMaster egg counting chamber. Only eggs in the vermiform and

infective larval stages were classed and counted as embryonated. The counting procedure

was repeated five times and the arithmetic mean was calculated. The infection dose was

then adjusted to 200 eggs/0.2 ml of final suspension. In the second and third repetitions,

the eggs of female worms harvested in the previous experimental run were used as

infection material and prepared in the same way. Therefore, age of the eggs at infection

was around 8 mo, 3 mo and 1 mo in the first, second and third repetition, respectively.

At the end of wk three, each feeding group was subdivided into an uninfected

control group (40% of birds) and an infected group (60%). The infected groups were

inoculated with 200 embryonated eggs of H. gallinarum per bird, which were administered

orally by a 5 cm esophageal cannula. Uninfected control birds were given 0.2 ml of an

aqueous placebo. Uninfected birds were left in their previous pens, whereas birds of each

infected group were placed in new pens within the same experimental stable. The birds did

not get any vaccination or anthelmintic treatment throughout the experimental period. The

stable was thoroughly disinfected at least 10 d before introducing the birds.

3.2.3. Chemical analyses of the diets

The compositions of the diets are same as given in Chapter 2. Nutrient contents of

the experimental diets are given in Table 1. Feed samples were taken regularly (at weeks 1,

6, 11) during each experimental repetition and were analyzed for dry matter (DM), crude

ash (CA), crude protein (CP), sugar, starch, and ether extract (EE) using standard methods

(Naumann and Bassler, 1997). Neutral and acid detergent fibre (NDF and ADF,

respectively) were analyzed according to Van Soest et al. (1991) and results are given

exclusive of ash. The metabolizable energy of the diets (MJ ME/kg DM) was calculated

(FMVO, 2008). Insoluble and soluble non-starch polysaccharides were measured using an

enzymatic test (Megazyme, 2007). Inulin was determined according to Naumann and

Bassler (1997).

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51


Item CON1 I-NSP2 S-NSP3 Analyzed composition

DM, g/kg 894 896 895 Nutrient, g/kg DM

Ash 53 51 53 CP 216 199 202 NDF 111 166 110 ADF 33 90 40 Ether extract 40 36 37 Starch 514 476 435 Insoluble NSP 103 172 104 Soluble NSP 20 23 25 Inulin - - 70

Calculated energy ME, MJ/kg DM4 13.64 12.58 12.21

1 Basal diet. 2 Insoluble non-starch polysaccharide supplemented diet = 1000 g CON plus 100 g pea bran. 3 Soluble non-starch polysaccharide supplemented diet = 1000 g CON plus 100 g chicory root meal. 4 ME, MJ/kg DM= [( g CP/kg DM x 0.01551) + (g CL/kg DM x 0.03431) + (g starch/kg DM x 0.01669) + (g

sugar/kg DM x 0.01301)]. Sugar contents of the diets were estimated based on sugar contents of the components.

3.2.4. Faecal sampling and post-mortem examinations

During the last four days of the last two repetitions, birds of the infected groups

were placed into individual cages for a 24 h period of faeces collection (12 birds d-1 group-

1). In the cages, the birds had free access to feed and water. Faeces excreted by each bird

accumulated in plastic bag-covered boxes underneath the cage. The total amount of faeces

per bird/day was weighed, transferred into a plastic cup and stirred thoroughly for at least 3

minutes to get a paste-like consistency that guaranteed a homogeneous distribution of the

eggs in the faeces. The number of eggs per gram of faeces (EPG) was quantified using a

modified McMaster counting technique (MAFF, 1986) and saturated NaCl as the flotation

liquid (density = 1.2 g/ml). The minimum detection level was 50 eggs / g faeces.

The birds were slaughtered after electrical stunning eight wk post-infection (p.i.) at

an age of 11 wk. Immediately after slaughter, the gastrointestinal tracts were removed and

the visceral organs were separated. Weights of liver (+gall bladder), pancreas, full caeca as

well as length of small intestine and each caecum were measured. Intact caeca from 10

birds per group (60 per repetition) were weighed, frozen and stored at -18 °C until

analyzed for volatile fatty acids (VFA).

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The caeca of the infected birds were further processed for parasitological

examinations to determine incidence and number of adult worms, as well as number of

larvae. The caeca were opened with scissors, the content was removed, and the caecal

walls were flushed to remove the worms. The surface moisture of the empty caeca was

removed using paper towel, and the empty caeca weight was determined. The caecal

content was flushed with low-pressure tap water through a sieve with a mesh aperture of

100 µm, and then transferred into Petri dishes to be examined by a stereomicroscope

(Gauly et al., 2008). The adult and immature worms were counted and the adults were

sexed. Average intact worm length was estimated by measuring 20 female and male worms

per bird. In cases of lower than 20 worms per sex were available, all the intact worms were

measured. Caeca samples of uninfected control birds (15-20% of each group) were also

processed to confirm infection free status of these groups. The caeca of the residual birds

of each uninfected group were pooled and checked for the presence of the nematode. A

total number of 475 birds and 287 faecal samples were examined for determination of

worm burdens and EPG, respectively.

3.2.5. pH and volatile fatty acids (VFA)

The frozen intact caeca were thawed at room temperature. The caecal content was

removed from the caeca, and 2 g were weighed and immediately afterwards suspended in

10 ml of distilled water. The sample was mixed using a vortex for around 5 seconds. The

pH was directly measured in this suspension using a pH electrode (InLab®Easy BNC, Fa.

Mettler Toledo) connected to a pH meter (GC 811, Fa, Schott). Thereafter, the suspension

was centrifuged at 2000 x g at room temperature for 20 min. Five ml of supernatant was

transferred to a glass tube, which contained 250µl international standard (4% methyl-

valeric acid in formic acid). The mixture was vortexed and two parallel sub-samples of 1.5

ml each were transferred to Eppendorf tubes. The parallels were centrifuged at 10000 x g

at room temperature for 10 min. After centrifugation, the samples were stored in a

refrigerator (+4°C) until gas chromatography.

For gas chromatography, a combined internal/external standard procedure was

applied using a packed column (10% Carbowax 20 MTPA SP1000 with 1% H3PO4 on

Chromosorb WAW, 80/100). Temperature for injection port was 170 °C, for detector

200 °C and for column 120 °C (isothermal). The gas chromatograph (Shimadzu GC 14B)

was equipped with a flame ionization detector (FID) and hydrogen was used as the carrier

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gas (Da Costa Gomez, 1999; Abel et al., 2002). The average of the parallels was used for

calculations.

The remaining caecal contents after sampling for VFA were used to determine dry

matter, crude ash and organic matter of the caecal contents.

3.2.6. Data management and statistical analyses

3.2.6.1. Parameter definitions, transformations and restrictions

Because the data of the infection variables positively skewed (Skewness > 0) and

showed non-normal (Kolmogorow-Smirnow, p<0.05) distributions, log-transformations

were employed. For this, individual infection parameters that described worm counts

(establishment rate, number of males, females, larvae, and total worm burden), EPG, total

number of eggs excreted per worm population of a bird within 24 h (eggs per day; EPD)

and female worm fecundity parameters were transformed by using the natural logarithm

(ln) function [ln(y)=Log(y)] to correct for heterogeneity of variance and to produce an

approximately normally distributed data set. Establishment rate was defined as the number

of worms per bird in relation to infection dose. Adult female worm fecundity was defined

based on both EPG (EPG per female worm) and EPD (EPD per female worm). Lengths of

the male and female worms, sex ratio (numbers of females / males) and the amount of

daily faeces of the infected birds were left untransformed.

In preliminary analyses (with fixed effect of repetition), no significant (P>0.05)

interaction effects of diet x infection x experimental repetitions on any of the performance

parameters (e.g., BW, ADG) were observed. This partly indicated reproducible effects of

diet and infections on the performance parameters over the repetitions. However, because

the experimental repetitions were performed in different periods of time, the effect of

repetition was included in the models as a random factor to ensure safe generalization of

the effects of the main factors (diet and infection) and to avoid any possible confounding

effect of time, in which the repetitions were performed, with any of the main factors.

3.2.6.2. Statistics

Statistical analyses were performed with SAS V9.1.3 (2010). Mortality data of the

3 repetitions were pooled, because the overall level was below 5 %. The effect of diet on

mortality in the pre-infection period (wk 1-3) was analyzed with logistic regression method

using the GENMOD procedure with the logit link function. The GENMOD procedure fits

the generalised linear models and suited for responses with binary outcomes (Kaps and

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Lamberson, 2004). For the infected period (wk 4-11) the mortality model was extended to

the effects of diet, infection and the interaction effect between diet and infection.

The effect of diet on worm-harboring birds as a proportion of experimentally

infected birds (incidence of infection) was separately analyzed for each repetition. The

differences between incidences of infection among the infected groups were analyzed with

Fisher’s exact test, performed for all possible pair-wise combinations of the three infected

groups.

For establishment rate, worm counts and nematode egg excretion variables, the

model included fixed effect of the diets and random effect of experimental repetitions

using the Proc MIXED. Data of VFA and visceral organ measurements were analyzed with

another mixed model that included fixed effects of diets, infection as well as interaction

effect of diet and infection. Effect of experimental repetition was included in the model as

random.

The model for the repeatedly measured performance variables (e.g. BW, feed:gain)

included fixed effects of diet, infection, experimental weeks (as age of birds) as well as all

possible interactions among these factors. The effect of experimental repetitions was

included in the model as a random factor. Furthermore, individual random effect of the

birds as the repeated subject within a repetition over the experimental weeks, was also

included in the model presented below.

Yijklm = µ + αi + βj + γk + (αβ)ij + (αγ)ik + (βγ)jk + (αβγ)ijk + al + bk(l)+ εijklm

Where;

Yijklm= observation.

µ = the overall mean.

αi = the effect of diet (i = 1,2,3).

βj = the effect of infection (j = 0,1).

γk = the effect of experimental weeks (k= 3-11 wk).

(αβ)ij = the interaction effect between diet and infection.

(αγ)ik = the interaction effect between diet and experimental weeks.

(βγ)jk = the interaction effect between infection and experimental weeks.

(αβγ)ijk = the interaction effect among diet, infection and experimental weeks.

al = random effect of repetitions (l=1,2,3).

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bk(l) = random effect of individual bird within repetition over the experimental weeks, the

variance between repeated measurements of the birds (subject) within a repetition.

εijklm = residual random error.

The repeatedly (weekly) measured variables were assumed to be correlated from one

measurement date to the next, and thus the covariance structure was set to be compound

symmetry.

3.2.6.3. Presentation of the results

After infection at the end of wk three, groups of uninfected and infected chickens

were kept according to a 3 x 2 factorial arrangement of treatments with diet and infection

as the main factors. Therefore, unless no significant interactions between the effects of diet

and infection were encountered, the data are presented as the main effects of diet and

infection. In case of significant interactions between diet and infection, the results are either

presented for the 6 single treatments or are mentioned correspondingly in the text. Tukey

adjusted post-hoc comparisons (Alpha= 0.05) were performed to either partition effects of

the main factors or to determine single group differences when a non interactive significant

main effect or a when significant interaction effect of the main factors was encountered,

respectively.

For the effects of the main experimental factors, the results are presented as least

square means (LSMEANS) with common pooled standard error (PSE). The PSE, calculated

from the output of mixed models for balanced data, was confirmed to be the same as for a

balanced data set that could be calculated from the output of GLM procedure as Root Mean

Square Error divided by the square root of the number of observations per treatment mean

as described by Pesti (1997). Because the numbers of observations in the groups were not

always balanced for certain data, the most conservative (the largest) standard error of

LSMEANS was represented as the pooled SE.

3.2.7. Ethical consideration


(200 eggs) given to each bird was within the range of the worm burdens that can be





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3.3. Results

3.3.1. Mortality, feed consumption and performance

Birds consuming S-NSP had a higher mortality rate (4.2%) than those on the I-NSP

diet (0.3%) during the pre-infection period from wk 1-3 (P=0.016). Most of the deaths

occurred in the first week. After infection (wk 4-11), infected birds had a mortality of 2.5%

compared to 0.4 % in uninfected birds (P=0.032). Most of the mortality cases were due to

cannibalism, which developed within a few weeks after infection mainly after introducing

birds to the new pens. During the pre-infection period (wk 1-3), birds receiving I-NSP and

S-NSP consumed 6 % more and roughly 1 % less feed, respectively, compared to CON

(Table 2). The coefficient of variation (CV) of feed consumption, calculated for repetitions

within feeding groups, was less than 3 %. Regarding the entire experimental period (wk 1-

11), birds on the I-NSP and S-NSP diets consumed 8 % and almost 2 % more than the

CON fed group and the CV within feeding groups was smaller than 5 %. Compared to

CON, the NSP-diets reduced the BW gain of birds and impaired the feed conversion rate

with I-NSP entailing a greater amount of feed per unit BW gain than S-NSP (P<0.001).

Infected birds consumed 1.6 % less feed and had a lower BW (P<0.001) accompanied by a

higher feed:gain ratio (P<0.001) than their uninfected counterparts.

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Table 2. Effects of diet and H. gallinarum infection on feed consumption, body weight (BW), and feed:gain ratio.

Diet1 H. gallinarum infection2 Item CON I-NSP S-NSP PSE 3 P-value Uninfected Infected PSE 3 P-value

Interaction P-value

Pre-infection period (wk 1-3) Feed consumption4, g/bird 336 356 332 - - no no no no no BW5, g 201a 192b 192b 8.778 0.001 no no no no no Feed:gain, g/g 2.10a 2.38b 2.20c 0.094 0.001 no no no no no

Entire period (wk 1-11) no no no no no Feed consumption4, g/bird 2910 3146 2964 - - 3030 2983 - - - BW6, g 989a 960b 954b 16.100 0.001 978A 957B 16.050 0.001 0.582 Feed:gain, g/g 3.30a 3.64b 3.50c 0.067 0.001 3.43A 3.53B 0.066 0.001 0.430

[(abc) or (AB)]: Different letters within each factor on the same line indicate differences (Tukey, p<0.05). no: no infection effect in the pre-infection period. 1 CON = basal diet; I-NSP = 1,000 g CON + 100 g pea bran; S-NSP = 1,000 g CON + 100 g chicory root meal. 2 Uninfected controls or infected with 200 eggs of Heterakis gallinarum. 3 Pooled SE. 4 Calculated from daily group consumptions, therefore no statistical comparison could be performed (-). 5 Body weight at the end of wk 3 of life. 6 Estimated from body weight of birds from wk 3-11 of life, presented as BW at the age of 11 wk.

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3.3.2. Incidence of infection, worm burdens and worm fecundity

In repetitions 1 and 3, feeding NSP led to higher incidences of infection than CON

(P<0.01), but no difference was observed in the second repetition. Almost all NSP fed

birds had at least one worm in all three repetitions. In CON fed birds, 88 %, 100% and 81

% were harboring worms in the first, second and third repetition, respectively. Birds on the

NSP-diets had higher establishment rate (P<0.001), number of larvae (P<0.009) and total

worm burden (P<0.001) than those receiving CON (Table 3). There was a trend for a lower

ratio of female to male worms in birds being fed S-NSP instead of CON (P=0.094). The

worm length was not influenced by diet (P>0.05).

Table 3. Effect of diet on establishment rate, average number of worms per bird, sex ratio

and length of worms in birds infected with Heterakis gallinarum (200 eggs/bird).

Diet1

Item CON I-NSP S-NSP PSE2 P-value, ≤

Establishment rate3, % 26.7a 46.9b 49.2b 11.959 0.001

Number of female worms3 26.6a 46.2a 47.6b 12.811 0.001

Number of male worms3 26.6a 45.9a 49.7b 11.481 0.001

Number of larvae3 0.15a 1.62b 1.03b 0.470 0.009

Total worm burden3 53.4a 93.7b 98.4b 23.917 0.001

Sex ratio, F/M 1.07 1.03 0.95 0.072 0.094

Female worm length, mm 11.28 11.47 11.37 0.302 0.111

Male worm length, mm 9.59 9.66 9.65 0.217 0.578

(ab): Values with no common letters within rows differ (Tukey, p<0.05). 1 CON = basal diet; I-NSP = 1,000 g CON + 100 g pea bran; S-NSP = 1,000 g CON + 100 g chicory root

meal 2 Pooled SE. 3 LSMEANS and PSE represent untransformed data, P-values are based on the transformed data.

The amount of faeces increased in the NSP-fed birds (P<0.001) irrespective of the

type of NSP (Table 4). The EPG, the EPD and EPD/female worm were elevated after

feeding S-NSP (P≤0.002), whereas I-NSP led to lower EPG/female worm (P<0.05). The

EPD increased in the sequence of CON < I-NSP < S-NSP (P<0.001).

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Table 4. Effect of diet on the amount of faeces, the excretion of nematode eggs and the

fecundity estimates of worms in birds infected with Heterakis gallinarum (200 eggs /

bird).*

Diet 1

Item CON I-NSP S-NSP PSE P-value, ≤

Faeces, g bird-1 d-1 28.26a 37.68b 36.43b 4.70 0.001

EPG2 449a 581a 780b 180.91 0.002

EPG / female worm3 13.2a 10.4b 14.9a 4.85 0.001

EPD4 12148a 19138b 26181c 3548.59 0.001

EPD / female worm5 321.4a 344.0a 490.8b 96.10 0.002

(*): LSMEANS and pooled standard error (PSE) represent untransformed data, P-values and multiple comparisons are based on transformed data. (abc): Values with no common letters within rows differ (Tukey, p<0.05). 1 CON= basal diet; I-NSP = 1,000 g CON + 100 g pea bran; S-NSP = 1,000 g CON + 100 g chicory root

meal. 2 Number of eggs per gram of faeces. 3 EPG based female worm fecundity: average number of eggs excreted per female worm through one gram of

faeces. 4 Number of eggs per day; total number of eggs excreted per worm population of a bird within 24 h. 5 EPD based female worm fecundity; number of eggs excreted per female worm within 24 h.

3.3.3. Visceral organ development

Both types of NSP increased the relative pancreas weight, the caecum length and

the empty caeca weight (P<0.001; Table 5), and, with the exception of relative pancreas

weight, S-NSP exerted a greater effect than I-NSP (P<0.05). The absolute liver weight was

lower in S-NSP fed birds than the CON fed birds (P=0.005), however relative weight of

liver to BW (hepato-somatic index) was not influenced by the diets (P=0.629). The small

intestine length and the full caeca weight were increased by S-NSP (P<0.05). Infection

reduced liver weight (P=0.005) and increased the relative pancreas weight (P=0.003) as

well as the full caeca weight (P=0.027). Empty caeca weight was also elevated by infection

(P<0.001). An interaction between diet and infection (P=0.039) revealed, that within each

feeding group infected animals had heavier empty caeca than the uninfected ones, but

uninfected S-NSP fed birds exceeded infected ones receiving CON and did not differ from

infected I-NSP-fed birds.

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Table 5. Effects of diet and H. gallinarum infection on the size of certain visceral organs.

Diet1 H. gallinarum infection2 CON I-NSP S-NSP PSE 3 P, ≤ - + PSE 3 P, ≤

Interaction P-value

Liver, g 18.0a 17.7ab 17.3b 0.499 0.005 17.9A 17.5B 0.494 0.005 0.849 HS-Index4, % (Liver/BW) 1.84 1.84 1.85 0.020 0.629 1.85 1.84 0.020 0.340 0.587 Pancreas, g 2.35 2.42 2.42 0.044 0.097 2.39 2.40 0.042 0.559 0.479 g Pancreas / 100 g BW 2.41a 2.53b 2.60b 0.087 0.001 2.47A 2.56B 0.086 0.003 0.244 Small int. length, cm 108.6a 108.5a 112.6b 1.505 0.001 110.2 109.6 1.482 0.362 0.669 Caecum length, cm 13.7a 14.1b 15.7c 0.083 0.001 14.4 14.5 0.074 0.242 0.075 Full caeca weight, g 6.42a 6.54a 9.52b 0.178 0.001 7.35A 7.64B 0.171 0.027 0.214 Empty caeca weight, g 2.46a 2.61b 3.29c 0.085 0.001 2.59A 2.98B 0.083 0.001 0.039

[(abc) or (AB)]: Different letters within each factor on the same line indicate differences (p<0.05). 1 CON = basal diet; I-NSP = 1,000 g CON + 100 g pea bran; S-NSP = 1,000 g CON + 100 g chicory root meal. 2 Uninfected controls (-) or infected with 200 eggs of Heterakis gallinarum (+). 3 Pooled SE. 4 HS-Index: Hepato-somatic index = liver/BW*100.

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3.3.4. Biochemical characteristics of the caeca

As shown in Table 6, percental DM of caecal contents was not influenced by diet

(P=0.246) but was decreased by infection (P<0.001). Feeding S-NSP decreased the

proportion of crude ash and increased organic matter in the DM of caecal contents

compared to feeding CON and I-NSP (P<0.001). Infection did not influence crude ash and

organic matter in the DM of caecal contents (P>0.05). Feeding S-NSP reduced intracaecal

pH and molar proportion of acetate and increased that of butyrate as well as the caecal

pools of individual and total VFA compared to CON and I-NSP (P<0.001). Infection

increased pH (P=0.002) accompanied by lower molar proportion of butyrate (P<0.001),

pools of acetate (P=0.003), butyrate (P<0.001) as well as the total VFA pool (P<0.001) of

the caecal contents. Significant interaction effects of diet and infection were observed for

the caecal propionate pool (P=0.014), which was smaller (P<0.05) in infected CON and I-

NSP fed birds and reached a similarly (P>0.05) high level in all the other groups (Figure).

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Table 6. Effects of diet and H. gallinarum infection on biochemical characteristics of the caeca. Diet1 H. gallinarum infection2 Item CON I-NSP S-NSP PSE 3 P, ≤ Inf. (-) Inf. (+) PSE 3 P, ≤

Interaction P-value

Dry matter (DM), % 17.48 17.63 18.19 0.398 0.246 18.42A 17.11B 0.350 0.001 0.589 Crude ash, (% of DM) 13.86a 13.15a 11.41b 0.377 0.001 12.65 12.97 0.339 0.317 0.373 Organic matter, (% of DM) 86.14a 86.85a 88.59b 0.377 0.001 87.35 87.03 0.339 0.317 0.373 pH 6.61a 6.59a 6.00b 0.153 0.001 6.32A 6.49B 0.151 0.002 0.210 VFA Molar ratios, %

Acetate 69a 71a 66b 1.301 0.001 68 69 1.229 0.091 0.785 Propionate 15 15 14 2.142 0.581 14 15 2.093 0.389 0.278 Butyrate 16a 15a 20b 1.219 0.001 18A 16B 1.183 0.001 0.212

VFA Pool, µmol4 Acetate 262.6a 247.0a 351.3b 20.769 0.001 312.8A 261.1B 18.945 0.003 0.116 Propionate 52.5a 47.5a 69.5b 8.416 0.001 62.0A 51.0B 8.210 0.003 0.014 Butyrate 61.0a 52.6a 108.9b 4.705 0.001 86.4A 62.0B 3.799 0.001 0.812 Total 376.2a 347.1a 529.7b 30.091 0.001 461.2A 374.2B 27.403 0.001 0.144

[(abc) or (AB)]: Different letters within each factor on the same line indicate significant differences (Tukey, p<0.05). 1 CON: Basal diet; I-NSP: Insoluble non-starch polysaccharide supplemented diet; S-NSP: Soluble non-starch polysaccharide supplemented diet. 2 Uninfected controls (-) or infected (+) with 200 eggs of Heterakis gallinarum. 3 Pooled SE. 4 Calculated as multiplication of VFA concentration by the total amount cecal digesta.

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aab

a

bcc

a

0

10

20

30

40

50

60

70

80

CON I-NSP S-NSP CON I-NSP S-NSP

Uninfected controls Infected

Pro

pio

nat

e, µ

mo

l / b

ird

Figure Interaction effect (P=0.014) of diet and infection on the propionate pool.

(abc): Different letters among the groups indicate significant differences (Tukey, p<0.05).

3.4. Discussion

The inclusion of pea bran and chicory root meal implied a nutrient dilution in I-

NSP and S-NSP diets compared to CON. However, because chickens are able to increase

their feed intake when a nutritionally diluted diet is offered (Forbes and Shariatmadari,

1994; Halle, 2002; Van Krimpen et al., 2007; Daş et al., 2010), the NSP fed birds could

have consumed similar amounts of basal mixture nutrients as the CON fed birds. In fact,

this was the case with I-NSP, whereas with S-NSP the increase in feed intake was not large

enough to reach a similar level of basal mixture intake compared to CON. In spite of these

differences in feed intake and the larger intestinal capacity of S-NSP fed birds, the NSP fed

birds developed lower BW than those on the CON.

The increased sizes and weights of empty caeca, pancreas and small intestine

length indicate that NSP feeding, with S-NSP in particular, have caused preferred

channeling of nutrients to the development of splanchnic tissues and the intestinal tract.

Increased relative pancreas in I-NSP and S-NSP fed birds indicates an adaptive response

induced either directly by the diet (Iji et al., 2001) or indirectly by the intestinal microbiota.

The longer small intestine in S-NSP fed birds indicates stimulated fermentation in the pre-

caecal intestine too. Greater caeca size has repeatedly been observed after feeding chicken

with NSP (Redig, 1989; Clench and Mathias, 1995; Jørgensen et al., 1996; Józefiak et al.,

2004). Fermentation of inulin in chicken has been reported to selectively support butyrate

Chapter-III


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producing microorganisms and to lower luminal pH (Marounek et al., 1999; Rehman et al.,

2008a,b). Volatile fatty acids and butyrate in particular are known to exert trophic effects

on the intestinal mucosa (Montagne et al., 2003). In the present study, the pools of VFA

and butyrate were increased and the pH was lowered in the caeca of birds on the chicory

root meal supplemented diet in agreement with the known effects of intestinal inulin

fermentation in chicken.

Dietary NSP intensified H. gallinarum infection in grower layers. Soluble NSP

enhanced fecundity of the female worms, which would contribute to a strongly

contaminated environment and create a higher risk for new or re-infections of birds under

field conditions. Soluble NSP also led to higher mortality of the birds in the pre- but not in

the post-infectional period from wk 4 onwards. This is in agreement with finding that the

adaptation of NSP-degrading enzyme activity requires time (Iji et al., 2001). Although

viscous S-NSP may stimulate proliferation of health impairing microorganisms in the

small intestine (Smits and Annison, 1996; Rehman et al., 2007), soluble NSP in the form

of inulin do not affect digesta viscosity (Schneeman, 1999). Infected birds had higher

mortality rate than their uninfected counterparts consuming the same diets, but most of the

deaths were due to cannibalism. Because in most cases, the intestines of victims were

partly or completely eaten by the pecking birds, it was not possible to investigate whether

cannibalism would relate to the infection status of the birds. Though a tendency toward

increased agonistic behavior in A. galli infected birds has been reported (Gauly et al.,

2007), cannibalism developed shortly after introducing the infected birds to the new pens

in the present study and this may also have contributed to the erratic behavior developed

during the establishment of a new social hierarchy in the infected groups.

Heterakis gallinarum is commonly regarded as a non-pathogenic nematode (Taylor

et al., 2007). However, the present study shows that the efficiency of feed utilization and

growth performance were impaired in infected birds independent of the type of diet. This

might have been caused by partial diversion of nutrients from growth to development of

immunity (Kyriazakis and Houdijk, 2006), and to support repair of damaged mucosal

structures of the intestine in infected birds (Hoste, 2001). Infection induced slight decrease

in feed intake of the birds might have also contributed to the retarded growth of the

infected birds.

The average establishment rate for the CON fed birds were similar to those

reported for chickens infected either with 100 eggs (Gauly et al. 2008) or with 3-9

eggs/bird (Fine, 1975). Insoluble and soluble NSP supplemented diets almost doubled the

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establishment rate. Establishment rate of H. gallinarum in a pheasant host system was

shown to be density dependent, i.e., the success rate of larvae developing to adult stage

decreases as the infection dose increases (Thompkins and Hudson, 1999). The same pattern

has also clearly been observed for A. galli in experimentally infected birds with different

infection doses (Permin et al., 1997). With regard to the density dependent characteristic of

the establishment rate, the great difference observed in the present study between CON and

NSP fed birds indicates that manifold favorable conditions were provided to the nematode

by feeding NSP. Although no difference between worm burdens of I-NSP and S-NSP fed

birds was observed, these two diets differed in their effects on the fecundity of the

nematode.

Because the nematode’s eggs are passed to the external environment throughout

caecal droppings (Fine, 1975), which are periodically excreted within a day (Clarke, 1979),

24 hour of collection of the final faeces, originating both from intestines and caeca seems

to be crucial for reliable egg quantification of this nematode. The EPG is widely used for

estimating the intensity of nematode infections in living host animals, it may however be

influenced by the amount of faeces, which was elevated in the NSP fed birds confirming

earlier results of others (Van der Klis et al., 1993; Jørgensen et al., 1996). Feeding I-NSP

led to higher worm burden, but did not increase EPG, and a lower EPG based fecundity

(EPG/female worm) was calculated in comparison to CON because of the greater amount

of faeces. Feeding S-NSP also led to greater worm burden and faeces amount, and EPG

was in addition increased without an effect on EPG based worm fecundity. Thus, the EPG

rendered unsatisfactory information about the actual infection intensity in the birds. Based

on EPD, I-NSP and particularly S-NSP increased worm egg excretion compared to CON

and the fecundity (EPD/female worm) was also elevated after feeding S-NSP. As shown in

the present study, the inclusion of the total daily amount of faeces for the calculation of

EPD eliminates dilution effect of faeces and provides more accurate information about the

actual infection status of the host animal as well as for the actual worm fecundity estimate

than EPG alone.

Infection increased caeca size possibly by a histotropic phase, in which the larvae

embed themselves into caecal tissue during the larval development as observed in A. galli

infection (Herd and McNaught, 1975). It has been shown that bacteria play an important

role for the establishment of H. gallinarum (Springer et al., 1970). The inulin supplied with

S-NSP in the present study can be regarded as prebiotic similar to pure inulin which has

been shown to increase bacteria counts and metabolic activity in turkeys (Juskiewicz et al.,

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2005) and in chickens (Rehman et al., 2008b). It can be assumed that the establishment and

fecundity of the bacteria feeder H. gallinarum (Bilgrami and Gaugler, 2004) was

stimulated by NSP fermenting caecal bacteria. An explanation for the reduced caecal VFA

pool observed in infected birds can only be found if the rates of VFA production and

utilization respectively are investigated in more detail. Undoubtedly, the nematode

benefited from the altered NSP dependent caecal environment resulting in higher

establishment rate and enhanced fecundity. However, when compared to the effects of S-

NSP on the gastrointestinal organ development and the parameters describing the

intracaecal environment (organic matter, pH, VFA), the effects of I-NSP were less

prominent than that of S-NSP. Enlarged caecal size (length and empty weight) induced by

feeding both NSP diets can be interpreted as an indication of a higher possibility (larger

space, less competition etc.) for establishment of larvae, while further altered caecal

environment due to feeding S-NSP corresponds well to the enhanced fecundity of the

nematode in the S-NSP fed birds.

Previous studies showed that insoluble NSP provide favorable conditions to

Oesophagostomum dentatum, the nodule worm of pigs, whereas S-NSP (inulin) was shown

to have adverse effects on this parasite (Petkevičious et al., 1997; 2001; 2003). Based on

these results, the authors have proposed inulin as a potential dewormer (Petkevičious et al.,

2003). The results reported from the pig studies are in agreement with the favorable effects

of I-NSP to H. gallinarum. However, in contrast to O. dentatum, the inulin rich S-NSP fed

in the present study also provided most favorable conditions to H. gallinarum. The

differences between responses of different worm species to the same substance is of

interest, and should further be investigated.

3.5. Conclusion

It is concluded that the pea bran and chicory root meal used as sources of insoluble

and soluble dietary NSP, respectively, provided favorable conditions for the establishment

of H. gallinarum in grower layers. Inulin rich chicory root meal additionally enhanced

fecundity of the nematode. Therefore, the two natural sources of insoluble and soluble NSP

offer no potential as protecting agents against H. gallinarum infections in chicken.

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References

Abel, Hj., Immig, I. Harman, E., 2002. Effect of adding caprylic and capric acid to grass

on fermentation characteristics during ensiling and in the artificial rumen system

RUSITEC. Anim. Feed Sci. Technol. 99, 65-72.



Clarke, P.L., 1979. Coccidial infection with Eimeria tenella and caecal defaecation in

chicks. Br. Poult. Sci. 20, 317-322.

Clench, M.H., Mathias, J.R.,1995. The avian cecum: a review. Wilson Bull. 107, 93-121.

Coop, R. L., Holmes, P.H., 1996. Nutrition and parasite interaction.

Int. J. Parasitol. 26, 951-962.


Da Costa Gomez, C., 1999. In-vitro-Untersuchungen zur reduktiven Acetogenese im

Pansen. Diss. agr. Göttingen, pp. 18–25.



Br. Poult. Sci. 50, 512-520.

Daş, G., Abel, H., Humburg, J., Schwarz, A., Rautenschlein, S., Breves, G., Gauly, M,

2009. Does Histomonas meleagridis affect establishment and development of

Heterakis gallinarum in chicken fed diets with different non-starch

polysaccharides? Proceedings of the XIV International Congress in Animal

Hygiene, Vol. 2: 573-576. Vechta, Germany.





Fine, P.E.M., 1975. Quantitative studies on the transmission of Parahistomonas wenrichi

by ova of Heterakis gallinarum. Parasitology. 70, 407-417.

Forbes, J.M., Shariatmadari, F., 1994. Diet selection by poultry. World. Poult. Sci J. 50, 7-

24.

Chapter-III


68

68




Gabrashanska, M., Teodorova, S.E., Galvez-Morros, M.M., Tsocheva-Gaytandzhieva, N.,

Mitov, M., Ermidou-Pollet, S., Pollet, S., 2004. Administration of Zn-Co-Mn basic

salt to chickens with ascaridiosis II. Sex ratio and microelement levels in Ascaridia

galli and in treated and untreated chickens. Parasitol. Res. 93, 242-247.

Gauly, M., Duss, C., Erhardt, G., 2007. Influence of Ascaridia galli infections and

anthelmintic treatments on the behaviour and social ranks of laying hens (Gallus

gallus domesticus). Vet. Parasitol. 146, 271-280.

Gauly, M., Kanan, A., Brandt, H., Weigend, S., Moors, E., Erhardt, G., 2008. Genetic

resistance to Heterakis gallinarum in two chicken layer lines following a single dose

of infection. Vet. Parasitol. 155, 74-79.

Halle, I., 2002. Einfluss einer gestaffelten Supplementierung von Lysin und Methionin

während der Aufzucht auf das Wachstum und auf die Leistungsmerkmale der Hennen

in der folgenden Legeperiode bei einer gestaffelten Protein- und Energieversorgung.

Arch. Geflugelkd. 66, 66-74.





Idi, A., Permin, A., Jensen, S.K., Murrel, K.D., 2007. Effect of a minor vitamin A

deficiency on the course of infection with A. galli (Schrank, 1788) and the

resistance of chickens. Helminthologia, 44, 3-9.

Iji, P.A., Saki, A.A., Tivey, D.R., 2001. Intestinal development and body growth of broiler

chicks on diets supplemented with non-starch polysaccharides


Jørgensen, H., Zhao, X-Q., Knudsen, K.E.B., Eggum, B.O., 1996. The influence of dietary

fibre source and level on the development of the gastrointestinal tract, digestibility

and energy metabolism in broiler chickens. Br. J. Nutr. 75, 379-395.



Chapter-III


69

69


ceca: a review. Anim. Feed Sci. Technol. 113, 1-15.



inulin. Arch. Geflugelkd. 69, 175-180.

Kaps, M., Lamberson, W. R., 2004. Biostatistics for Animal Science, pp: 394-412. CABI

Publishing, Wallingford, U.K.



MAFF, 1986. Manual Veterinary Parasitological Laboratory Techniques, Ministry of

Agriculture, Fisheries and Food, Reference Book 418, 3rd edition, HMSO, London.

Marounek, M., Suchorska, O., Savka, O., 1999. Effect of substrate and feed antibiotics on

in vitro production of volatile fatty acids and methane in cecal contents of chickens.


Maurer, V., Amsler, Z., Perler, E., Heckendorn, F., 2009. Poultry litter as a source of

gastrointestinal helminth infections. Vet. Parasitol. 161, 255-260.


Avian Dis. 49, 462-476.





non-ruminant animals. Anim. Feed Sci. Technol, 108, 95-117.




prevalence of gastrointestinal helminths in different poultry production systems. Br.

Poult. Sci. 40, 439-443.

Permin, A., Bojesen, M., Nansen, P., Bisgaard, M., Frandsen, F., Pearman, M., 1997.

Ascaridia galli populations in chickens following single infections with different

dose levels. Parasitol.Res. 83, 614-617.

Chapter-III


70

70

Pesti, G.M., 1997. Pooled standard error??. Poultry Sci. 76, 1624.










68.

Puellen, U., Chat, S., Moors, E., Gauly, M., 2008. The role of preparation technique,

culture media and incubation time for embryonation of Heterakis gallinarum eggs.

Deutsche Tierarztliche Wochenschrift, 115:30-33.

Redig, P., 1989. The avian ceca: obligate combustion chambers or facultative afterburners?

The conditioning influence of diet. J. Exp. Zool. Suppl. 3, 66-69.



61, 319-335.

Rehman, H., Hellweg, P., Taras, D., Zentek, J., 2008a. Effects of dietary inulin on the



Rehman, H., Böhm, J., Zentek, J., 2008b. Effects of differentially fermentable


Anim. Physiol. Anim. Nutr. 92, 471-480.





Nutr. 129, 1424-1427.

Chapter-III


71

71

Smits, C.H.M., Annison, G., 1996. Non-starch plant polysaccharides in broiler nutrition –

towards a physiological valid approach to their determination. World. Poultry Sci. J.

52, 203-221.



383-392.

Stear, M.J., Doligalska, M., Donskow-Schmelter, K., 2007. Alternatives to anthelmintics

for the control of nematodes in livestock. Parasitology. 134, 139-151.



1.

Thompkins, D.M., Hudson, P.J., 1999. Regulation of nematode fecundity in the ring-

necked pheasant (Phasianus colchicus): not just density dependence. Parasitology.

118, 417-423.






eating time and performance of hens in early lay. Br. Poult. Sci. 48, 389-398.



Sci. 74, 3583-3597.





Chapter-IV

Effects of NSP on A. galli

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CHAPTER -IV Effects of dietary non-starch polysaccharides in Ascaridia galli-infected grower layers

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Effects of dietary non-starch polysaccharides in Ascaridia galli-infected grower layers

Abstract

This study examined if the establishment and fecundity of Ascaridia galli in chicken can

be controlled by dietary non-starch polysaccharides (NSP). One-day-old female layer

chicks (N=604) were fed ad libitum either a basal diet (CON) or CON plus pea bran rich in

insoluble NSP (I-NSP), or CON plus chicory root meal as a source of inulin rich soluble

NSP (S-NSP) for 11 wk in a three times repeated experiment. At the end of wk 3, each

feeding group was subdivided into an uninfected and an infected group of birds, the latter

being inoculated with 250 embryonated eggs of A. galli, respectively. The birds were

slaughtered 8 wk post infection and their worm burdens and the faecal nematode egg

excretion were determined. Volatile fatty acids (VFA) and pH were determined in caeca

contents.

Both NSP diets, particularly S-NSP, increased incidence of infection (P<0.05) and worm

burden of birds (P<0.001), but the development (length) and fecundity of the nematode

remained unaffected (P>0.05). A. galli infection caused a less efficient feed utilization for

body weight gain (BWG, P=0.013) resulting in lower body weights (P<0.001) irrespective

of type of diet consumed. NSP-fed birds, particularly those on I-NSP, consumed more feed

per unit BWG and showed retarded body weight development (P<0.001) compared to birds

receiving CON. Birds receiving I-NSP had higher body weights than those consuming S-

NSP (P<0.05). Intracaecal pH was lowered by feeding S-NSP (P<0.05) independent of A.

galli infection (P=0.223). Both NSP diets increased total VFA pool size (P<0.001), S-NSP

exerting a greater effect than I-NSP (P<0.05). Infected birds had smaller total VFA pool

size than their uninfected counterparts consuming the corresponding diets (P=0.028). S-

NSP also led to higher weights of splanchnic tissues and intestinal tract (P<0.05). These

effects were less pronounced in I-NSP fed birds.

The results suggest that insoluble and soluble dietary NSP retard growth

performance, alter gastrointestinal environment and lead to higher weights of splanchnic

tissues associated with an elevated establishment of A. galli in grower laying hens. It is

concluded that the NSP used in this study had no potential for controlling A. galli infection

in the birds.

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Keywords: Non-starch polysaccharides; Ascaridia galli; pea bran; chicory root; inulin;

chicken.

4.1. Introduction

Dietary non-starch polysaccharides (NSP) are fermentatively degraded in the distal

sections of the gastrointestinal tract (Englyst, 1989; Schneeman, 1999). Apart from certain

anti-nutritive effects of NSP mainly associated with increased digesta viscosity (Daenicke

et al., 1999; Francesch and Brufau, 2004; Daenicke et al., 2009), dietary NSP may support

animal welfare and health. Van Krimpen et al. (2008) reported that hens fed diets high in

insoluble NSP increased time spent for eating and reduced aggressive pecking behaviours.

Coarsely ground NSP supplemented diets may also stimulate the development of the

gizzard suggesting improved digestive functioning (Van Krimpen et al., 2009). The soluble

NSP inulin is known for its prebiotic effect (Schneeman, 1999) and inulin-dependent

stimulation of metabolic activity of beneficial intestinal bacteria has been reported for

turkeys (Juskiewicz et al., 2005) and chickens (Rehman et al., 2007; Rehman et al., 2008a).

Similar to some other EU countries the number of chickens kept in floor husbandry

is increasing in Germany (ZMP, 2008), but in these systems the birds are in close contact

with their faeces, allowing nematodes to complete their life cycles. The prevalence of

common poultry nematodes has been reported to be higher in outdoor/floor husbandry

systems than in conventional ones (Permin et al., 1999; Kaufmann and Gauly, 2009). The

common fowl parasite, Ascaridia galli is a widespread nematode of chickens that resides in

the small intestine. There is evidence that dietary NSP may interact with parasites of the

host animals. Daenicke et al. (2009) showed that feeding viscous NSP favored the

development of A. galli. In pigs, the type of dietary NSP has been shown to affect the

establishment, development and fecundity of common nematodes (Petkevičius et al., 1997;

2001; 2003). The effects of NSP on nematode infections are assumed to be mainly

attributable to alterations of digesta characteristics and intestinal microbial fermentation.

We hypothesized that the establishment and fecundity of A. galli in chicken is affected by

dietary NSP. The objective of the present study was to investigate the effects of low or

highly fermentable NSP on the establishment and fecundity of the nematode as well as on

the performance of grower layers experimentally infected with A. galli in order to examine

if the nematode infection can be controlled by dietary NSP.

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4.2. Material and methods

In three repetitions, 604 female Lohmann Selected Leghorn (LSL) chicks were used

in the time interval from July 2007 to July 2008. The chicks were purchased from a

commercial hatchery as one-day-old bird. Within each repetition, the chicks were weighed

together and randomly divided into three feeding groups. Each feeding group was fed ad

libitum until wk 11 of life one of the following pelleted diets: basal diet (CON), CON plus

insoluble NSP (I-NSP), and CON plus soluble NSP (S-NSP) (Table 1). Insoluble NSP

were supplied by mixing on air dry-basis one kg CON with 100 g pea bran (Exafine 500,

Socode, Belgium). For S-NSP, one kg CON was mixed with 100 g chicory root meal

(Fibrofos 60, Socode, Belgium). Daily feed consumption was determined per group

throughout the experimental weeks. Drinking water was offered ad libitum.

Until wk three, each feeding group was kept in a pen scattered with wood shavings.

The litter was replaced once (wk 1-3) or twice (wk 4-11) a week. Room temperature was

gradually decreased from 34 C on the first day (d) to 26 C in wk 3 and thereafter

decreased by 2-3C per wk, ending at 18-20 C from wk six onwards. A 24 h lighting

period was maintained for the first two days and was then reduced to 16 h/d at the end of

the first week. By wk eight, it was reduced to 12h/d and subsequently maintained until the

end of the experiment. At the end of wk three, the birds were marked with wing tags and

individual body weights (BW) were taken for the first time and thereafter at weekly

intervals.

2.2. Infection material and experimental A. galli infections

The infection material was prepared at the Department of Animal Sciences,

University of Göttingen, Germany. Adult female A. galli worms harvested from the

intestines of naturally infected chickens were used as the source of eggs. Preparation

techniques for the infection material (egg harvest, embryonation procedures etc.) were

performed as described in a previous study (Daş et al., 2010a) in details. On the infection

day, number of eggs/ml suspension was determined and the infection dose was adjusted to

250 eggs/0.2 ml of final suspension. Eggs only in the vermiform and infective larval stages

were classed and counted as embryonated. Three sub-groups of birds, each for one feeding

group, were infected at an age of three wk using a five cm oesophageal cannula. The

remaining three groups of uninfected control birds were given 0.2 ml of an aqueous 0.1%

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solution (w/v) of potassium dichromate as placebo. Average number of birds inoculated

with eggs per each feeding group in each repetition ranged from 30 to 35.

In the second and third repetitions, mixed batches of eggs from female worms

harvested in the preceding repetition as well as eggs from worms of naturally infected field

chickens were used as the infection material and prepared in the same way. Average age of

the eggs (after embryonation) on infection days was around three, five and one mo in the

first, second and third repetition, respectively.

After inoculation of the eggs, birds of three uninfected control groups were left in

their previous pens, whereas birds of each infected group were placed in new pens within

the same experimental stable equipped with six pens. The birds did not get any vaccination

or anthelmintic treatment throughout the experimental period. The stable was thoroughly

cleaned and disinfected at least two wk before introducing the birds.

2.3. Slaughter process, faecal samples and post-mortem examinations

All the birds were slaughtered after electrical stunning 8 wk post-inoculation (p.i.)

at an age of 11 wk. The slaughtering was accomplished within three h on the day of

slaughter. Individual faecal samples were collected during the slaughter process either as

freshly dropped faeces or - if available - directly from the colon. The individual faecal

samples were examined for estimating number of eggs per gram of faeces (EPG) using a

modified McMaster counting technique with a sensitivity of 50 eggs/g faeces (MAFF,

1986).

Immediately after slaughtering, the gastrointestinal tracts were removed and the

visceral organs were separated. The small intestines of the infected birds were opened

longitudinally with scissors. The contents were flushed with tap water through a sieve with

a mesh aperture of 100 µm, and then transferred into one or more Petri dishes, depending

on the amount of content. Thereafter, the incidence and number of adult worms and larvae

were determined using a stereomicroscope. The adults were sexed and a maximum number

of 10 (range 1-10) intact adult female and male worms per bird were measured for length

(Gauly et al., 2002). Small intestines from uninfected control birds (15-20% of each group)

were also processed to verify infection-free status of these groups. The remaining small

intestines from the uninfected control birds were opened and macroscopically checked for

the presence of adult worms.

Weights of liver (+gall bladder), pancreas, full caeca as well as length of small

intestine and each caecum were measured. In the last two repetitions, caeca from 10 birds

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per group (60 per repetition.) were weighed and frozen stored at -18 °C until analyzed for

volatile fatty acids (VFA).

2.4. pH and volatile fatty acids (VFA)

The frozen caeca were thawed at room temperature for pH measurement and VFA

analyses. The caecal content was removed from the caeca and the amount was quantified.

A two g sample of caecal content was weighed and suspended in 10 ml of distilled water.

The sample was mixed using a vortex for around five seconds. The pH was directly

measured in this suspension using a pH electrode (InLab®Easy BNC, Fa. Mettler Toledo)

connected to a pH meter (GC 811, Fa, Schott). Thereafter, the suspension was centrifuged

at 2000 x g at room temperature for 20 min. Five ml of supernatant was transferred to a

glass tube, which contained 250µl international standard (4% methyl-valeric acid in formic

acid). The mixture was vortexed and two parallel sub-samples of 1.5 ml each were

transferred to sample tubes. The tubes were centrifuged at 10.000 x g at room temperature

for 10 min. After centrifugation, the samples were stored in a refrigerator (+4°C) until gas

chromatography.

For gas chromatography, a combined internal/external standard procedure was

applied using a packed column (10% Carbowax 20 MTPA SP1000 with 1% H3PO4 on

Chromosorb WAW, 80/100). Injection port temperature was 170 °C, for detector 200 °C

and for column 120 °C (isothermal). The gas chromatograph (Shimadzu GC 14B) was

equipped with a flame ionization detector (FID) and hydrogen was used as the carrier gas

(Da Costa Gomez, 1999; Abel et al., 2002). The average of two parallel analyses for each

sample was used for calculations.

In the last repetition, the remaining caecal contents after sampling for VFA were

used to determine dry matter and crude ash contents in order to calculate the organic

substance in the caeca samples.

2.5. Chemical analyses of the diets

Feed samples were taken regularly during each experimental repetition and were

analyzed for dry matter (DM), crude ash (CA), crude protein (CP), sugar, starch, and ether

extract (EE) using standard methods (Naumann and Bassler, 1997). Neutral and acid

detergent fibre (NDF and ADF, respectively) were analyzed according to Van Soest et al.

(1991). The metabolizable energy of the diets (MJ ME/kg DM) was calculated (FMVO,

2008). Insoluble and soluble NSP were measured using an enzymatic test (Megazyme,

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2007). Inulin content of S-NSP diet was determined according to Naumann and Bassler

(1997). The composition and analyzed nutrient contents of the experimental diets are given

in Table 1.


Item CON I-NSP S-NSP Analyzed composition

DM, g/kg 898 900 900 Nutrients, g/kg DM

Ash 55 52 55 CP 218 198 203 NDF 113 173 122 ADF 34 92 36 Ether extract 40 36 37 Starch 486 446 423 Insoluble NSP 103 177 102 Soluble NSP 20 24 26 Inulin - - 77

ME, MJ/kg DM1 13.21 12.06 12.02 1 The metabolizable energy of the diets: calculated according to the formula given by the German regulations

for complete poultry feed mixtures (FMVO, 2008). ME, MJ/kg DM= [( g CP x 0.01551) + (g CL x 0.03431) + (g starch x 0.01669) + (g sugar x 0.01301)]. Sugar contents of the diets were estimated based on sugar contents of the components.

4.2.6. Data management and statistical analyses

4.2.6.1. Parameter definitions, transformations and restrictions

Because the data of the infection variables positively skewed (Skewness > 0) and

showed non-normal (Kolmogorow-Smirnow, p<0.05) distributions, log-transformations

were employed. For this, individual infection parameters that described worm counts

(establishment rate, number of males, females, larvae, and total worm burden), number of

eggs per gram of faeces (EPG) and female worm fecundity parameters were transformed

by using the natural logarithm (ln) function [ln(y)=Log(y+1)] to correct for heterogeneity

of variance and to produce an approximately normally distributed data set. Establishment

rate was defined as the number of worms per bird in relation to infection dose. Worm egg

excretion was quantified as the number of eggs per gram of faeces (EPG) in birds that had

had a faecal sample. Fecundity of adult female worms was defined as EPG per female

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worm. Lengths of the male and female worms and sex ratio (numbers of females / males)

were left untransformed.

Because the experimental repetitions were performed at different periods of time

and we used different batches of worm eggs, the effect of repetition was included in the

models as a random factor to ensure safe generalization (with a cost of elevated standard

errors) for the effects of the main experimental factors and to avoid any possible

confounding effect of time, in which the repetitions were performed, with any of the main

factors.

2.6.2. Statistics

Effects of the diets on the incidence of A. galli infection (proportion of worm-

harbouring birds to the experimentally infected birds) were analyzed using GENMOD

procedure of SAS (2010) with a logit link function. The GENMOD procedure fits the

generalised linear models and suited for responses with binary outcomes (Kaps and

Lamberson, 2004). The model included effects of diet and effect of repetition. Because

there was no significant interaction effect between diets and repetitions the following

reduced model was used (I).

(I) ij = log [pij / (1 - pij)]= m + i + rj

i= diets; CON, I-NSP, S-NSP

j= repetitions; 1, 2, 3

where;

pij= the proportion of infected birds on diet i and repetition j

m= the overall mean of the proportion on the logarithmic scale

i = the effect of diet i

rj= the effect of repetition j

Body weight (BW) and feed utilization (feed:gain) data were analyzed both for pre-

infectional (1-3 wk) and for the entire period (1-11 wk). The model for the performance

parameters in pre-infectional period included fixed effect of diets, random effect of

repetitions and the residual error (II). This model was also used to analyze establishment

rate, worm counts, worm length and egg excretion data.

(II) Yijk = µ + αi + aj +εijk

where;

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Yijk= observation.



aj = random effect of repetition (j =1,2,3).

εijk = residual random error.

Organ measurements and VFA data were analyzed with the following mixed model (III).

(III) Yijkl = µ + αi + βj + (αβ)ij + ak +εijkl

where;

Yijkl= observation.




(αβ)ij = the interaction effect between diet and infection (ij = 1-6).

ak = random effect of repetition (k* =1,2,3).

εijkl = residual random error.

*: (k=1, 2) for the VFA data.

The model for the repeatedly measured performance variables (BW, feed:gain) in

the entire period included fixed effects of diet, infection, experimental weeks (as age of

birds) as well as all possible interactions among these factors. The effect of experimental

repetitions was included in the model as a random factor. Furthermore, individual random

effect of the birds as the repeated subject within a repetition over the experimental weeks,

was also included in the model presented below (IV).

(IV) Yijklm = µ + αi + βj + γk + (αβ)ij + (αγ)ik + (βγ)jk + (αβγ)ijk + al + bk(l)+ εijklm

Where;

Yijklm= observation.



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γk = the effect of experimental weeks (k= 3-11 wk).

(αβ)ij = the interaction effect between diet and infection.

(αγ)ik = the interaction effect between diet and experimental weeks.

(βγ)jk = the interaction effect between infection and experimental weeks.

(αβγ)ijk = the interaction effect among diet, infection and experimental weeks.

al = random effect of repetition (l=1,2,3).

bk(l) = random effect of individual bird within repetition over the experimental weeks, the

variance between repeated measurements of the birds (subject) within a repetition.

εijklm = residual random error.

2.6.3. Presentation of the results

After infection at the end of wk 3, groups of uninfected and infected chickens were

kept according to a 3 x 2 factorial arrangement of treatments with diet and infection as the

main factors. Therefore, unless no significant interactions between the effects of diet and

infection were encountered, the data are presented as the main effects of diet and infection.

In case of significant interactions between diet and infection, the results are either presented

for the 6 single treatments or are mentioned correspondingly in the text. Tukey adjusted

post-hoc comparisons (Alpha< 0.05) were performed to either partition effects of the main

factors or to determine single group differences when a non interactive significant main

effect or when a significant interaction effect of the main factors was encountered,

respectively.

For the effects of the main experimental factors, the results are presented as least

square means (LSMEANS) with common pooled standard error (PSE). The PSE, calculated

from the output of mixed models for balanced data, was confirmed to be the same as for a

balanced data set that could be calculated from the output of GLM procedure as Root Mean

Square Error divided by the square root of the number of observations per treatment mean

as described by Pesti (1997). Because the numbers of observations in the groups were not

always balanced for certain data, the most conservative (the largest) standard error of

LSMEANS is represented as the pooled SE.

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2.7. Ethical consideration


given to each bird (250 eggs) was within the range of the worm burdens that can be





4.3. Results

4.3.1. Mortality, feed consumption and growth performance

Infected birds did not show clinical signs of infection and the overall mortality rate

was low. Uninfected control birds were free of infection as confirmed by microscopic and

macroscopic examination of the small intestines. Because the mortality rate in the pre-

infectional period was low (1.2 %) no statistical comparison among the feeding groups was

done. Birds consuming S-NSP had a slightly higher mortality (2.1 %) than those on the

CON (0.9 %) or the I-NSP diet (0.6 %). In the post-inoculation period (wk 4-11), infected

and uninfected birds had similar low mortality rates (3 %).

During the pre-infection period, birds receiving I-NSP and S-NSP consumed

roughly 4.5 % and 1.7 % more feed, respectively, than those being fed CON (Table 2). The

coefficients of variation (CV) for feed consumption calculated within feeding group over 3

repetitions in the pre-infectional period, were less than 6 %. Birds consuming I-NSP had a

higher feed:gain ratio in comparison to CON and S-SNP fed birds (P<0.05). Both NSP-

diets reduced the BW of birds in comparison to CON (P<0.001).

In the entire experimental period, birds on the I-NSP and S-NSP diets consumed 8

% and 2 % more than the CON fed birds. The CVs within each feeding groups were lower

than 5 %. Compared to CON, birds on the NSP-diets consumed more feed per unit body

weight gain (BWG, P<0.001) and I-NSP caused higher feed intake than S-NSP (P<0.05).

Both NSP diets led to retarded body weight development (P<0.001) with S-NSP entailing a

stronger negative effect than I-NSP (P<0.05). Within feeding groups, infected birds

consumed almost the same amount of feed as the uninfected birds whereas body weight of

the birds was impaired irrespective of type diet (P<0.001) resulting in lowered efficiency

of feed utilization (P=0.013).

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Table 2. Effects of diet and A. galli infection on feed consumption, body weight development (BW), and feed:gain ratio (N=581).

Diet1 A. galli infection2 Period/Item CON I-NSP S-NSP PSE 3 P, ≤ Uninfected Infected PSE 3 P-value

Interaction P-value

Pre-infection (wk 1-3) Feed consumption4, g/bird 336 351 330 - - no no no no no BW5, g 202a 197b 196b 6.833 0.001 no no no no no Feed:gain, g/g 2.08a 2.24b 2.12a 0.116 0.001 no no no no no

Entire-period(wk 1-11) Feed consumption4, g/bird 2899 3129 2963 - - 3006 2990 - - BW6, g 981a 953b 939c 11.048 0.001 972A 942B 10.936 0.001 0.296 Feed:gain, g/g 3.09a 3.43c 3.30b 0.078 0.001 3.22A 3.33B 0.070 0.013 0.084

[(abc) or (AB)]: Different letters within each factor on the same line indicate differences (p<0.05). no: no infection effect in the pre-infection period. 1 CON = basal diet; I-NSP = 1,000 g CON + 100 g pea bran; S-NSP = 1,000 g CON + 100 g chicory root meal. 2 Uninfected controls or infected with 250 eggs of A. galli. 3 Pooled SE. 4 Estimated from daily group consumptions. 5 Body weight at the end of wk 3 of life. 6 Body weight at the end of wk 11 wk (i.e., 8 wk p.i).

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4.3.2. Incidence of infection, worm burdens and worm fecundity

There was a significant effect of diet on the incidence of infection (P<0.001). As

shown in Table 3, it was higher with S-SNP than with CON and I-NSP (P<0.05). Feeding

I-NSP also tended to increase incidence of infection when compared with CON (P=0.078).

Birds on the NSP-diets had higher establishment rates (P<0.001), higher numbers of

female (P=0.003) and male worms (P=0.021) as well as higher total worm burden

(P<0.001) than those receiving CON. The number of larvae tended (P=0.060) to be higher

in S-NSP than in CON fed birds. Sex ratio, worm length, EPG and female worm fecundity

remained unaffected by type of diet (P>0.05).

Table 3. Effect of diet on establishment rate, average number of worms per bird, sex ratio,

length and egg excretion parameters of worms in birds infected with Ascaridia galli (250

eggs / bird).

Diet1

Item CON I-NSP S-NSP PSE2 P-value, ≤

Incidence, % (N=286) 57.0a* 68.4a* 89.0b - 0.001

Establishment rate3, % 0.91a 1.34b 1.36b 0.382 0.001

Number of female worms3 0.74a 1.21b 1.19b 0.403 0.003

Number of male worms3 1.12a 1.55b 1.50b 0.624 0.021

Number of larvae3 0.38 0.56 0.70 0.124 0.060

Total worm burden3 2.27a 3.34b 3.40b 0.955 0.001

Sex ratio, F/M 0.69 0.67 0.80 0.138 0.687

Female worm length, cm 7.13 7.35 7.46 0.377 0.572

Male worm length, cm 5.15 5.45 5.59 0.120 0.122

Eggs per gram of faeces (EPG)3 359 319 223 177 0.363

Fecundity (EPG / female worm)3 210 91 125 60 0.576 ab: Values with no common letters within rows differ (Tukey, p<0.05). *: Values sharing the sign tend to differ (P=0.078). 1 CON = basal diet; I-NSP = 1,000 g CON + 100 g pea bran; S-NSP = 1,000 g CON + 100 g chicory root

meal 2 PSE: Pooled standard error. 3 LSMEANS and PSE represent untransformed data, P-values are based on the transformed data.

4.3.3. Visceral organ development

No significant interaction effect was observed between diet and infection on any of

the organ measurements (P>0.05). The absolute weights of liver and pancreas were not

affected by the type of diet (P>0.05; Table 4), but the NSP diets increased the relative

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weights of both organs (P<0.05). The small intestine length, the caecum length and the full

caeca weight were increased by S-NSP (P<0.05).

A. galli infection increased, the absolute pancreas weight and the length and weight

of caeca as well as the relative liver and pancreas weights (P<0.05). The liver weight

tended to be higher in the infected birds (P=0.058). Infected birds had shorter small

intestines than their uninfected counterparts receiving the same diets (P=0.007).

4.3.4. Biochemical parameters of the caecal content

As shown in Table 5, both NSP diets increased the amount of caeca content

(P<0.001) with S-NSP exerting a greater effect than I-NSP (P<0.05). Feeding S-NSP

increased the proportions of dry matter in the caecal contents (P<0.05). The proportion of

organic matter was increased (P<0.05) at the expense of crude ash (P<0.05) in the caecal

content of S-NSP fed birds. Infection did not influence the proportions of organic matter

and ash (P>0.05). The intracaecal pH was lower with feeding S-NSP (P<0.05) and

remained unaffected by the infection (P>0.05).

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Table 4. Effects of diet and A. galli infection on the size of visceral organs (N=567).

Diet1 A. galli infection2 CON I-NSP S-NSP PSE 3 P, ≤ - + PSE 3 P, ≤

Interaction P-value

Liver, g 17.8 18.0 17.7 0.363 0.472 17.7 18.0 0.356 0.058 0.208 HS-Index4, % (Liver/BW) 1.82a 1.89b 1.90b 0.065 0.001 1.83A 1.91B 0.065 0.001 0.221 Pancreas, g 2.36 2.38 2.38 0.076 0.726 2.33A 2.41B 0.075 0.001 0.957 g Pancreas / 100 g BW 2.42a 2.51b 2.55b 0.117 0.001 2.42A 2.57B 0.116 0.001 0.902 Small int. length, cm 105.0a 104.9a 108.9b 1.148 0.001 107.0A 105.5B 1.121 0.007 0.379 Caecum length, cm 13.56a 13.81a 15.12b 0.267 0.001 13.89A 14.43B 0.264 0.001 0.239 Full caeca weight, g 6.33a 6.59a 8.89b 0.168 0.001 7.16 7.38 0.158 0.075 0.085

[(abc) or (AB)]: Different letters within each factor on the same line indicate differences (p<0.05). 1 CON = basal diet; I-NSP = 1,000 g CON + 100 g pea bran; S-NSP = 1,000 g CON + 100 g chicory root meal. 2 Uninfected controls (-) or infected with 250 eggs of A. galli (+). 3 Pooled SE. 4 HS-Index: Hepato-somatic index = liver/BW*100.

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Table 5 Effects of diet and A. galli infection on biochemical parameters of caecal content*.

Diet1 A. galli infection2 Item CON I-NSP S-NSP PSE 3 P, ≤ Inf. (-) Inf. (+) PSE 3 P, ≤

Interaction P-value

Caecal content, g 3.49a 4.13b 5.12c 0.182 0.001 4.44 4.04 0.148 0.060 0.052 Dry matter (DM), % 16.6a 17.1a 21.0b 0.484 0.001 19.0A 17.5B 0.392 0.006 0.127 Ash, % (of DM) 14.0a 13.4a 9.9b 0.390 0.001 12.7 12.1 0.308 0.181 0.074 Organic matter, % (of DM) 85.8a 86.6a 90.1b 0.307 0.001 87.3 87.9 0.308 0.181 0.074 pH 5.97a 6.03a 5.35b 0.053 0.001 5.82 5.75 0.042 0.223 0.079 VFA pool, µmol4

Acetate 223a 275b 296b 15.272 0.001 282A 248B 12.961 0.035 0.040 Propionate 18a 29b 34b 1.966 0.001 27 27 1.619 0.763 0.630 Butyrate 64a 73a 114b 9.342 0.001 92A 76B 8.863 0.011 0.713 Total 305a 377b 445c 25.016 0.001 401A 350B 22.233 0.028 0.140

*: N=120 for ph and VFA data, for the remaining variables N=60. [(abc) or (AB)]: Different letters within each factor on the same line indicate significant differences (Tukey, p<0.05). 1 CON: Basal diet; I-NSP: Insoluble non-starch polysaccharide supplemented diet; S-NSP: Soluble non-starch polysaccharide supplemented diet. 2 Uninfected controls (-) or infected (+) with 250 eggs of A. galli. 3 Pooled SE. 4 Calculated as multiplication of VFA concentration by the total amount cecal digesta.

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Pool size of acetate was influenced by interaction between diet and infection

(Figure; P=0.040). Infected birds on CON had lower pool size of acetate than all the other

infected or uninfected birds on any diet (P<0.05). Pool size of propionate was increased by

NSP diets (P<0.05; Table 5) and remained unaffected by infection (P=0.763). Butyrate

pool size was increased with S-NSP (P<0.05) and decreased by A. galli infection

(P=0.011). Both NSP supplemented diets increased the total VFA pool size (P<0.001), S-

NSP exerting a greater effect than I-NSP (P<0.05). Infected birds had smaller total VFA

pool size than their uninfected birds consuming the corresponding diets (P=0.028).

Figure Pool size of acetate in the caeca as influenced by interaction between diet and infection (P=0.040; N=120).

4.4. Discussion

As the study was designed, the authors assumed that the three ad libitum offered

experimental diets would supply chickens with similar amounts of essential nutrients and

energy either via CON alone or with the CON proportions of the I-NSP and S-NSP diets.

This assumption was based on expected compensatory feed consumption of birds receiving

the nutritionally diluted NSP diets (Forbes and Shariatmadari, 1994; Halle, 2002; Van

Krimpen et al., 2007). According to this concept, infection with A. galli should clearly be

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testable as affected by insoluble and soluble NSP. In fact, birds receiving I-NSP instead of

CON increased feed intake by 8 % thereby almost balancing feed dilution brought about by

9% pea bran. In S-NSP fed birds, the increase in feed intake was not large enough to

compensate feed dilution and their proportionate CON intake was approximately 7 %

lower than that of birds on the pure CON diet. Therefore, differences between birds in their

proportionate CON consumption may have additionally influenced the results.

In spite of almost compensatory feed intake, I-NSP fed birds had lower final BW

than those on the pure CON diet, indicating that consumption of I-NSP may have been

associated with additional metabolic costs. In S-NSP fed birds, the reduced final BW may

have at least partially resulted from lower proportionate intake of CON. Inulin, which was

fed as a source of soluble NSP, is known to increase digesta bulk (Schneeman, 1999) and

feed intake of S-NSP fed birds might have been limited by the capacity of the

gastrointestinal tract.

Dietary insoluble and soluble NSP favored A. galli infection in terms of higher

incidence of infection and worm burdens. The absence of clinical signs of Ascaridiosis and

low mortality may be explained by the low worm burdens. The average worm burden of

the infected birds was, however, in the range observed by others applying similar or the

same infection doses (Riedel and Ackert, 1951; Gauly et al., 2001; Marcos-Atxutegi et al.,

2009; Daenicke et al., 2009; Daş et al., 2010a). A. galli infection did not influence feed

intake of the birds, but caused a less efficient utilization of feed for BWG. This indicates

an altered allocation of nutrients from growth to defense reactions against the parasite

infection (Kyriazakis and Houdijk, 2006). Diverted allocation of nutrients may include

repair of damaged mucosal tissues during the histotropic phase of the nematode (Herd and

Mcnaught, 1975) and the acquisition as well as the development of immunity (Marcos-

Atxutegi et al., 2009), which may require high metabolic inputs (Colditz, 2008). A. galli

infection may also influence digestion and absorption of nutrients by reducing proteolytic

enzyme activity in the jejunum (Hurwitz et al. (1972a) being associated with an increased

recycling of nitrogen (Hurwitz et al., 1972b). Reduced metabolizability of energy and

lowered nitrogen retention has been reported for A. galli infected chickens (Walker and

Farrell, 1976).

Both NSP supplemented diets increased incidence of infection and worm burdens

of the birds but the development (length) and fecundity of the nematode remained

unaffected. Thus, dietary NSP appear to support larvae establishment. The average female

worm lengths measured in the present study are in agreement with earlier reports

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(Abdelqader et al., 2007; Daenicke et al., 2009; Marcos-Atxutegi et al., 2009). The average

worm fecundity (EPG/female) was slightly lower for the worms of the NSP fed birds and

this may have resulted from increased faeces amounts as has been reported for chicken

submitted to NSP feeding (Van der Klis et al., 1993; Jørgensen et al., 1996). In a parallel

experiment with Heterakis gallinarum, NSP fed birds produced roughly 33% more faeces

than birds on a NSP-unsupplemented diet. Such increase would lead to lower EPG levels

and the EPG based fecundity estimates may be underestimated even at significantly

elevated worm burden (Daş et al., 2010b), In the present investigation, no difference in

EPG between CON and NSP fed birds was observed because the latter presumably

excreted larger daily amounts of faeces. A more precise evaluation of female worm

fecundity could be achieved by referring worm egg excretion on total daily faeces

excretion of the birds.

Soluble NSP present in certain cereals like e.g., rye, barley, triticale and wheat may

exert anti-nutritive effects in poultry through increased intestinal viscosity (Francesch and

Brufau, 2004; Józefiak et al., 2004; Daenicke et al., 2009). Daenicke et al. (2009) showed

that soluble NSP-caused increased viscosity favored the development of A. galli and

elevated nematode egg excretion. Soluble NSP in the form of inulin do not affect digesta

viscosity (Schneeman, 1999), but, as observed in the present study and in agreement with

others (Montagne et al., 2003), led to elevated weights of splanchnic tissues as well as

increased sizes of gastrointestinal tract sections. The enlarged caeca can be related to

increased microbial activity. The praecaecal section was obviously also exposed to

stimulated fermentation, as indicated by increased small intestine size in S-NSP fed birds.

The lowered pH and the greater pools of VFA in caeca contents are further indications for

an inulin-induced stimulation of intestinal fermentative activity. Consistent with previous

observations (Marounek et al., 1999; Rehman et al., 2008a, b), fermentation of inulin was

associated with high proportions of butyrate, which in turn can be used by colonocytes as a

source of energy for epithelial cell proliferation, thus supporting the enlargement of

intestinal tract sections (Montagne et al., 2003).

Increased worm burdens with NSP feeding can be related to an altered

gastrointestinal environment in the birds. However, apart from greater effects of S-NSP

than I-NSP on enlargement of gastrointestinal organs and caecal VFA pool size, A. galli

infection also increased caeca size and the relative proportions of liver and pancreas.

Increased proportion of liver has been reported in A. galli infected birds and may indicate

Chapter-IV


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91

liver dysfunction and disturbance in carbohydrate metabolism in the infected birds

(Ramadan and Abou Znada, 1991).

Energy diluted diets have been suggested for laying hens in order to benefit from

compensatorily increased feed intake, thereby balancing nutrient intake of the animals

under the condition of organic farming (Sundrum et al., 2005; Van de Weerd et al., 2009).

Considering the availability of organic feedstuffs, such energy dilutions will primarily be

achieved by fibre, i.e. NSP rich feed components. The results of the present investigation,

showing infection supporting effects of dietary NSP, and the inherently increased risk of

obligatory outdoor sytems for parasitic infections (Permin et al., 1999; Thamsborg et al.,

1999; Fossum et al., 2009) suggest particular measures of precaution for organic poultry

husbrandy in order to protect animals against nematode-related health risks.

4.5. Conclusion

Insoluble and soluble dietary NSP retard growth performance, alter gastrointestinal

environment and lead to higher weights of splanchnic tissues being associated with an

elevated establishment of A. galli in grower laying hens. These observations may be

particularly relevant for poultry husbandry in organic farming, where relatively fibre, i.e.

NSP rich feeding is recommended and where animals with obligatory outdoor access are

inherently exposed to high nematode infection risks.

References

Abdelqader, A., Gauly, M., Wollny, C.B.A., 2007. Response of two breeds of chickens to

Ascaridia galli infections from two geographic sources. Vet. Parasitol. 145, 176-180.

Abel, Hj., Immig, I. Harman, E., 2002. Effect of adding caprylic and capric acid to grass

on fermentation characteristics during ensiling and in the artificial rumen system

RUSITEC. Anim. Feed Sci. Tech. 99, 65-72.

Colditz, I.G., 2008. Six costs of immunity to gastrointestinal nematode infections. Parasite

Immunol. 30, 63-70.

Da Costa Gomez, C., 1999. In-vitro-Untersuchungen zur reduktiven Acetogenese im

Pansen. Diss. agr. Göttingen, pp. 18–25.

Daenicke, S., Dusel, G., Jeroch, H., Kluge, H., 1999. Factors affecting efficiency of NSP-

degrading enzymes in rations for pigs and poultry. Agribiol. Res, 52, 1-24.

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Brit. Poultry Sci. 50, 512-520.

Daş, G., Kaufmann, F., Abel, H., Gauly, M., 2010 (a) .Effect of extra dietary lysine in


Daş, G., Abel, H., Gauly, M., 2010 (b). Faeces amount influences eggs per gram of faeces

counts in chicken. Tierarztliche Praxis (G) Abstracts, 4, V-49. DVG –

Fachgruppentagung Parasitologie und parasitäre Krankheiten, München 07.- 09.

Juli 2010.


Sci. Tech. 23, 27-42.

Forbes, J.M., Shariatmadari, F., 1994. Diet selection by poultry. World. Poult. Sci. J. 50, 7-

24.



doi:10.1186/1751-0147-51-3.

Francesch, M., Brufau, J., 2004. Nutritional factors affecting excreta/litter moisture and

quality. World. Poult. Sci. J. 60, 64-75.




Gauly, M., Bauer, C., Mertens, C., Erhardt, G., 2001. Effect and repeatability of Ascaridia

galli egg output in cockerels following a single low dose infection. Vet. Parasitol. 96,

301-307.

Gauly, M., Bauer, C., Preisinger, R., Erhardt, G., 2002. Genetic differences of Ascaridia

galli egg output in laying hens following a single dose infection. Vet. Parasitol. 103,

99-107.







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93



Hurwitz, S., Shamir, N., Bar, A., 1972 (a). Effect of Ascaridia galli on lumen activity of

enzymes in the intestine of chicks. Poultry Sci. 51, 1462-1463.

Hurwitz, S., Shamir, N., Bar, A., 1972 (b). Protein digestion and absorption in the chick:

effect of Ascaridia galli. Am. J. Clin. Nutr. 25, 311-316.



and energy metabolism in broiler chickens. Brit. J. Nutr. 75, 379-395.


ceca: a review. Anim. Feed Sci. Tech. 113, 1-15.



inulin. Arch. Geflugelkd. 69, 175-180.

Kaps, M., Lamberson, W. R., 2004. Biostatistics for Animal Science, pp: 394-412. CABI

Publishing, Wallingford, U.K.

Kaufmann, F., Gauly, M., 2009. Prevalence and burden of helminths in laying hens kept in

free range systems. Proceedings of the XIV International Society for Animal

Hygiene Congress, Vol. 2: 557-560. Vechta, Germany.



MAFF, 1986. Manual Veterinary Parasitological Laboratory Techniques, Ministry of

Agriculture, Fisheries and Food, Reference Book 418, 3rd edition, HMSO, London.

Marcos-Atxutegi, C., Gandolfi, B., Arangüena, T., Sepúlveda, R., Arévalo, M., Simón, F.,

2009. Antibody and inflammatory responses in laying hens with experimental

primary infections of Ascaridia galli. Vet. Parasitol. 161, 69-75.

Marounek, M., Suchorska, O., Savka, O., 1999. Effect of substrate and feed antibiotics on

in vitro production of volatile fatty acids and methane in cecal contents of chickens.

Anim. Feed Sci. Tech. 80, 223-230.



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94



non-ruminant animals. Anim. Feed Sci. Tech. 108, 95-117.






Pesti, G.M., 1997. Pooled standard error??. Poultry Sci. 76, 1624.


and sugar beet fibre on Oesophagostomum dentatum in pigs. Parasitology, 127, 61-

68.



dentatum in the intestinal tract of pigs. Parasitology, 123, 315-324.




Parasitology, 114, 555-568.

Ramadan, H.H., Abou Znada, N.Y., 1991. Some pathological and biochemical studies on

experimental Ascaridiasis in chickens. Nahrung-Food. 35, 71-84.

Rehman, H., Böhm, J., Zentek, J., 2008b. Effects of differentially fermentable


Anim. Physiol. AN. N. 92, 471-480.

Rehman, H., Hellweg, P., Taras, D., Zentek, J., 2008a. Effects of dietary inulin on the





61, 319-335.

Chapter-IV


95

95





Nutr. 129, 1424-1427.

Sundrum, A., Schneider, K., Richter, U., 2005. Possibilities and limitations of protein

supply in organic poultry and pig production. Final Project Report EEC 2092/91

(Organic) Revision no. D 4.1 (Part 1), Department of Animal Nutrition and Animal

Health, University of Kassel, Witzenhausen, Germany.



186.

Van de Weerd, H.A., Keatinge, R., Roderick, S., 2009. A review of key health-related

welfare issues in organic poultry production. World. Poult. Sci. J. 65, 649-684.



gastrointestinal tract of broilers. Brit. Poultry Sci. 34, 971-983.



eating time and performance of hens in early lay. Brit. Poultry Sci. 48, 389-398.


Verstegen, M.W.A., 2008. Low dietary energy concentration, High nonstarch

polysaccharide concentration, and coarse particle sizes of nonstarch polysaccharides

affect the behavior of feather-pecking-prone laying hens. Poultry Sci. 87, 485-496.


Verstegen, M.W.A., 2009. Effects of nutrient dilution and nonstarch polysaccharide

concentration in rearing and laying diets on eating behavior and feather damage of

rearing and laying hens. Poultry Sci. 88, 759-773.



Sci. 74, 3583-3597.

Chapter-IV


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63-77.





Zentrale Mark- und Preisberichtstelle (ZMP) GmbH, 2008. Marktbilanz, Eier und Geflügel

2008. Bonn, 213 pp.

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5. General discussions

5.1. Body weight development and feed intake

As observed in all three experiments, both, the NSP-supplemented diets and the

parasitic infections caused a retarded body weight development of the birds. Due to high

content of insoluble and presumably undegradable fibre, which is additionally known to

accelerate digesta passage rate (Hetland et al., 2004), total organic matter digestibility of I-

NSP was presumably lower than that of CON and S-NSP. In spite of compensatory feed

intake, I-NSP fed birds had lower final BW than those on the pure CON-diet, indicating

that consumption of I-NSP may have been associated with additional metabolic costs. In S-

NSP fed birds, the reduced final body weights may have resulted at least partially from

lower proportionate intake of basal diet (CON).

The NSP-supplemented diets used in this study were diluted as to the contents of

energy and nutrients. When the study was designed, it was assumed that the three ad lib.-

offered experimental diets would supply chickens with similar amounts of energy and

nutrients either via CON alone or with the CON proportions of I-NSP and S-NSP. This

assumption was based on expected compensatory feed consumption of birds receiving the

nutritionally diluted NSP-diets (Forbes and Shariatmadari, 1994; Halle, 2002; Van

Krimpen et al., 2007). According to this concept, birds on the NSP-supplemented diets

would increase their feed consumption to reach a similar level of energy and nutrient

intake as the CON-fed birds. In fact, birds receiving I-NSP instead of CON increased feed

intake by 8 % thereby almost balancing feed dilution brought about by 9% pea bran. In S-

NSP-fed birds, the increase in feed intake was not large enough (2%) to compensate feed

dilution and their proportionate CON-intake was approximately 7 % lower than that of

birds on the pure CON-diet. These results have repeatedly been observed in the H.

gallinarum- (Chapter 3) and the A. galli- experiments (Chapter 4). In the histomonas-

contaminated H. gallinarum-experiment (Chapter 2) the increases were slightly lower

(6.2% and 1% for I-NSP- and S-NSP-fed birds, respectively) than in the prementioned two

experiments. In spite of the larger intestinal capacity of the S-NSP-fed birds, these birds

were not able to increase their feed intake similarly to those of the I-NSP-fed birds. The

lower nutrient intake in the S-NSP-fed birds may therefore have additionally influenced all

the results reported in this study. Although effects of feeding additional insoluble fibre on

infections and performance of the birds have clearly been tested by comparing the I-NSP-

and CON-feeding groups, the lower nutrient intake in the S-NSP-fed birds might, to some

Chapter-V

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extent, have interfered with the effects of soluble NSP supplied with chicory root meal.

However, these effects can be considered as total diet effects that include the effects of

soluble NSP-supplementation and lower nutrient intake.

What may have been the reason for the differences between feed consumption of I-

NSP- und S-NSP-fed birds, and why were the latter obviously unable to reach a similar

level of intake as the former? Increased intestinal viscosity due to dietary viscous NSP has

been reported to reduce feed intake of chickens (Van der Klis et al., 1993). However,

soluble NSP in the form of inulin do not affect digesta viscosity (Schneeman, 1999). These

results were also confirmed by parallel studies focusing on digestibility of the NSP diets

conducted within the same frame of this study. Humburg (2010) observed that neither I-

NSP nor S-NSP was associated with increased intestinal viscosity. In fact, this can be

expected because the main feedstuffs used in the experimental diets that may increase

intestinal viscosity were barley and wheat. Their proportions in the NSP-supplemented

diets, after dilution with either pea bran or chicory root, are reduced so that even a lower

intestinal viscosity associated with viscous-NSP supplementation could result. Therefore, it

can be assumed that lower feed intake in the S-NSP-fed birds must have been associated

with factors other than viscosity. Inulin, which was fed as a source of soluble NSP, is

known to increase digesta bulk (Schneeman, 1999) and feed intake of S-NSP-fed birds

might have been limited by the capacity of the gastrointestinal tract. On the other hand,

fermentation of NSP may increase heat production which may negatively influence feed

intake of the birds. Jørgensen et al. (1996) showed that fermentation of pea fibre

supplemented diets increased heat production of broilers relative to ME by 3-6%. As

shown by lowered intracaecal pH and elevated pool size of VFA in the present

investigation, the inulin supplemented S-NSP-diet intensified intestinal fermentation more

than the I-NSP-diet. Therefore, it may be assumed that intestinal fermentation increased

heat production, which contributed to the lower feed intake of the S-NSP-fed birds.

In the H. gallinarum-experiments, infection associated reductions in feed intake of

the birds were observed. The infection-induced reduction in voluntary feed intake was

stronger with histomonas-contamintated H. gallinarum-infection than in the pure H.

gallinarum-infection. In the A. galli-experiments there was no difference in feed intake

between infected and uninfected birds. This is in agreement with a previous study (Daş et

al., 2010) showing that A. galli-infection does not influence feed intake of the birds on a

nutritionally balanced diet. Anorexia, as observed in case of many parasitic infections, is

known as one of the main factors affecting performance of infected animals (Kyriazakis et

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100

al. 1998; Crop and Kyriazakis, 1999; Walkden-Brown and Kahn, 2002). The degree of

anorexia may be affected by the species of parasite and its site of infection as well as level

of infection and immune responses of the host animal (Knox et al., 2006). A direct

comparison between intensity of H. gallinarum- and A. galli-infections based on number of

worms may not be a valid approach. However, number of worms may indicate potential

parasitic stimulants that interact with the immune system. Lymphoid structures play an

important role in the avian immune system. Yegani and Korver (2008) reported that the

chicken’s foregut is relatively poor in lymphoid follicles, but they are numerously present

in the hindguts, particularly in the caeca. An intense immune stimulation induced by H.

meleagridis and H. gallinarum, might therefore, have contributed to the decreased feed

intake of the infected birds.

Adverse effects of parasitic infections on performance of the birds are not limited to

reduction in feed intake, but also to a less efficient utilization of feed for BWG. Immune

responses to gastrointestinal antigenic stimulations can negatively affect feed utilization

efficiency, are energetically expensive, and divert nutrients away from production (Yegani

and Korver, 2008). Parasitic infection-induced less efficient feed utilization often indicates

a diverted allocation of nutrients from growth to defense reactions against the parasite

infection (Kyriazakis and Houdijk, 2006) including repair of damaged mucosal tissues

during the histotropic phase of the nematodes (Herd and Mcnaught, 1975) and the

acquisition as well as the development of immunity (Marcos-Atxutegi et al., 2009). These

reactions may require high metabolic inputs (Colditz, 2008). A. galli infection may also

influence digestion and absorption of nutrients by reducing proteolytic enzyme activity in

the jejunum (Hurwitz et al., 1972a) being associated with an increased recycling of

nitrogen (Hurwitz et al., 1972b). Reduced metabolizability of energy and lowered nitrogen

retention has been reported for A. galli infected chickens (Walker and Farrell, 1976).

Heterakis gallinarum is commonly regarded as a non-pathogenic nematode (Taylor et al.,

2007). Although infection with this parasite is very common, effects of the infection on the

digestion processes are not known. However, the present study shows that the efficiency of

feed utilization and growth performance were impaired in H. meleagridis and/or H.

gallinarum infected birds irrespective of the type of diet, and thus indicating negative

effects of the caecal parasitic infections on the overall digestion and absorption processes.

5.2. Parasitic infections intensified by the dietary NSP

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Feeding NSP-supplemented diets did not only influence mono-infections with H.

gallinarum or A. galli, but also interactions between H. meleagridis and H. gallinarum. In

all parasitic infections, dietary NSP intensified infections as indicated by higher worm

burdens of the birds. It was shown that insoluble and soluble NSP differ in their effects on

the parasitic infections. The differences were more obvious with H. gallinarum- than with

A. galli-infections. Although both NSP supplemented diets favored the establishment of H.

gallinarum, S-NSP additionally enhanced female worm fecundity. Thus, it can be expected

that feeding S-NSP will aggravate environmental contamination with the excreted H.

gallinarum eggs and create an increased risk for new infections and re-infections of the

birds, if kept in the field. The type of dietary NSP also played an important role in the

interaction between H. meleagridis and H. gallinarum. Feeding S-NSP resulted in lower

incidence and total worm burden than I-NSP in case of histomonas contaminated H

gallinarum infection, whereas this diet led to highest worm burden when histomonas was

eliminated.

To our knowledge, effects of dietary NSP on parasitic infections in poultry have

only scarcely been investigated. Daenicke et al. (2009) showed that feeding viscous NSP

favored the development of A. galli and caused a higher level of egg excretion but did not

influence worm burden of growing chickens. In contrast to chickens, NSP-feeding has

extensively been investigated in pigs. Studies with the pig whipworm, Trichuris suis

showed that a dietary supplementation of 6 % inulin did not influence the establishment

rate but retarded worm growth (Thomsen et al., 2006), whereas 16 % inulin

supplementation decreased establishment, egg excretion and female worm fecundity

(Petkevičius et al., 2007) when compared with oat hull meal as source of insoluble NSP.

Similar results have also been reported for the pig nodule worm Oesophagostomum

dentatum. It has repeatedly been shown that lignin rich diets or supplementations of

insoluble NSP provided favorable conditions for the establishment of the nodule worm,

whereas inulin supplemented diets had a profound devorming effect (Petkevičius et al.,

1997; 2001; 2003). In another study with intestinally cannulated pigs, the same authors

showed that intracaecal infusion of SCFA and lactic acid reduced worm counts by 92%,

suggesting that the anti-parasitic effect of inulin is mediated through its metabolic products

such as increased SCFA and lactic acid concentration resulting from intestinal microbial

activity (Petkevičius et al., 2004).

As shown by our own as well as by the studies in pigs, the effects of NSP on

nematode infections can mainly be attributed to alterations of digesta characteristics and

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102

intestinal microbial fermentation. The favorable effects of I-NSP for H. gallinarum- and A.

galli- infections in birds are in agreement with the reported results of the pig studies.

However, in contrast to the effects of inulin in association with T. suis- and O. dentatum-

infections, the inulin rich S-NSP diet in the present study also provided most favorable

conditions for H. gallinarum- and A. galli-infections. The responses of different worm

species residing in their specific hosts to the same substance (e.g., inulin) remain further to

be investigated.

5.3. Technical issues in determination of nematode egg excretion

Estimations of gastrointestinal nematode infection intensity in the living animals

widely rely on the quantification of nematode egg concentration in host animal faeces, and

mostly expressed as number of eggs per gram of faeces (EPG). Although H. gallinarum

has been subject to many studies, egg excretion of this nematode has scarcely been

performed (Fine, 1975). This is high probably because of technical difficulties in sampling

of the right faecal material. Collection of daily faeces from infected birds did not only

provide a valid approach for quantification of the egg excretion of this nematode, but also

proved a weakness of EPG. In the mono H. gallinarum-experiment (Chapter 3) we

demonstrated that EPG is not a valid indicator for estimating infection intensity in case of

the total amount of faeces are expected to differ among groups to be compared. EPG

rendered unsatisfactory information about the actual infection intensity and the worm

fecundity in the birds on the NSP diets. The inclusion of the total daily amount of faeces

for the calculation of daily total number of excreted eggs (EPD) eliminates dilution effect

of faeces and provides more accurate information about the actual infection status of the

host animal as well as for the actual worm fecundity estimates than EPG alone.

5.4. General conclusions

Histomonas meleagridis does not only harm the definitive host, but also its vector,

Heterakis gallinarum. Insoluble and soluble NSP supplemented diets favor H. gallinarum

infection while S-NSP additionally intensifies histomonas infection, which then impairs

the establishment and development of H. gallinarum. The pea bran and chicory root meal

used as sources of insoluble and soluble dietary NSP, respectively, favored the

establishment of histomonas-free H. gallinarum in grower layers. Chicory root meal

Chapter-V

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103

additionally enhanced the fecundity of this nematode. Insoluble and soluble dietary NSP

retard the growth, alter the gastrointestinal environment, lead to higher weights of

splanchnic tissues and elevate the establishment of A. galli in grower layers. The

enhancing effects of the dietary NSP on the nematode infections are presumably caused by

an altered gastrointestinal environment, with S-NSP being more affective than I-NSP. It is

concluded that the two natural sources of insoluble and soluble NSP offer no potential as

protecting agents against parasitic infections in grower layers. Therefore, suitable measures

of precaution should be applied to production systems particularly prone to gastrointestinal

parasitic infections and where diets with relatively high NSP-contents are fed.

References

Colditz, I.G., 2008. Six costs of immunity to gastrointestinal nematode infections. Parasite

Immunol. 30, 63-70.




Br. Poult. Sci. 50, 512-520.



Fine, P.E.M., 1975. Quantitative studies on the transmission of Parahistomonas wenrichi

by ova of Heterakis gallinarum. Parasitology. 70, 407-417.

Forbes, J.M., Shariatmadari, F., 1994. Diet selection by poultry. World. Poult. Sci J. 50, 7-

24.







Hetland, H., M. Choct, and B. Svihus. 2004. Role of insoluble non-starch polysaccharides

in poultry nutrition. World. Poult. Sci. J. 60, 415–422.

Chapter-V

General Discussions

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104



Humburg, J., 2010. Einflüsse von Nich-Stärke-Polysacchariden des Futters und von

Nematodeninfektionen auf Verdauungsprozesse bei wachsenden Junghennen. Diss.

agr. Göttingen.



and energy metabolism in broiler chickens. Br. J. Nutr. 75, 379-395.

Knox, M.R., Torres-Acosta, J.F.J., Aguilar-Caballero, A.J. 2006. Exploiting the effect of

dietary supplementation of small ruminants on resilience and resistance against

gastrointestinal nematodes. Vet. Parasitol. 139, 385-393.



Kyriazakis, I., Tolkamp, B.J., Hutchings, M. R., 1998. Towards a functional explanation

for the occurrence of anorexia during parasitic infections. Anim. Behav. 56, 265-274.

Marcos-Atxutegi, C., Gandolfi, B., Arangüena, T., Sepúlveda, R., Arévalo, M., Simón, F.,

2009. Antibody and inflammatory responses in laying hens with experimental

primary infections of Ascaridia galli. Vet. Parasitol. 161, 69-75.





Petkevičius, S., Murrell, K.D., Knudsen, K.E.B., Jorgensen, H., Roepstorff, A., Laue, A.,

Wachmann, H., 2004. Effects of short-chain fatty acids and lactic acids on survival of

Oesophagostomum dentatum in pigs. Vet. Parasitol. 122, 293-301.

Petkevičius, S., Thomsen, L.E., Knudsen, K.E.B., Murrell, K.D., Roepstorff, A., Boes, J.,

2007. The effect of inulin on new and on patent infections of Trichuris suis in

growing pigs. Parasitology. 134, 121-127.




Chapter-V

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105



68.


Nutr. 129, 1424-1427.



1.

Thomsen, L.E., Petkevičius, S., Knudsen, K.E.B., Roepstorff, A., 2006. The influence of

dietary carbohydrates on experimental infection with Trichuris suis in pigs.







eating time and performance of hens in early lay. Br. Poult. Sci. 48, 389-398.

Walkden-Brown, S.W., Kahn, L.P., 2002. Nutritional modulation of resistance and

resilience to gastrointestinal nematode infection. Asian-Austr. J. Anim. Sci. 15, 912–

924.



63-77.

Yegani, M., Korver, D.R., 2008. Factors affecting intestinal health in poultry. Poultry Sci.

87, 2052-2063.

Zusammenfassung

106

106

ZUSAMMENFASSUNG

Ziel der vorliegenden Arbeit war es, die Hypothese zu prüfen, ob leicht lösliche oder

unlösliche Nicht-Stärke-Polysaccharide (NSP) einen Einfluss auf eine Heterakis gallinarum

beziehungsweise Ascaridia galli Infektion bei Aufzuchthennen haben. Da Heterakis

gallinarum als Vektor für Histomonas meleagridis, dem Erreger der Schwarzkopfkrankheit,

fungiert, wurde weiterhin untersucht, ob ein NSP-angereichertes Futter Einfluss auf

Wechselwirkungen dieser beiden Parasiten hat.

Die Untersuchungen wurden zwischen 2007 und 2010 am Department für

Nutztierwissenschaften der Universität Göttingen durchgeführt. Es kamen drei verschiedene

Fütterungsvarianten zum Einsatz. Der Nährstoffgehalt und die umsetzbare Energie der

Grundmischung (CON) entsprachen den Vorgaben und Empfehlungen für

Aufzuchtlegehennen. Erbsenschalen und Zichorienwurzelmehl dienten als Komponenten für

unlösliche bzw. lösliche NSP. In der Fütterungsvariante I-NSP wurden pro kg CON 100 g

Erbsenschalen und in der Fütterungsvariante S-NSP pro kg CON 100 g Zichorienwurzelmehl

zugesetzt. Die drei Versuchsmischungen CON, I-NSP und S-NSP wurden pelletiert.

Im ersten Experiment sollte der Einfluss eines mit NSP angereicherten Futtermittels

auf die Wechselwirkung zwischen H. gallinarum und H. meleagridis untersucht werden.

Dieses Experiment beinhaltete eine prophylaktische, über das Tränkesystem applizierte

Behandlung der Hälfte der Tiere mit Dimetridazol (0,05%) gegen H. meleagridis. Die durch

diesen Versuch gewonnenen histomonadenfreien weiblichen H. gallinarum Würmer dienten

als Infektionsmaterial für die anderen Versuchsdurchgänge mit H. gallinarum.

Der Einfluss von NSP des Futters auf H. gallinarum beziehungsweise A. galli wurde

in zwei aufeinanderfolgenden Versuchen mit jeweils 3 Wiederholungen pro

Nematodenspecies, untersucht. In jedem Durchgang wurden 3 Fütterungsgruppen gebildet,

die mit je einer der Versuchsmischungen bis zu einem Alter von 3 Wochen gefüttert wurden.

Nach 3 Wochen wurden die Tiere individuell mit Flügelmarken gekennzeichnet und

gewogen. Jede Fütterungsgruppe wurde nochmals in eine infizierte und eine nicht-infizierte

Gruppe unterteilt. Die infizierten Versuchsgruppen wurden jeweils mit je 200 embryonierten

H. gallinarum- beziehungsweise 250 embryonierten A. galli- Eiern künstlich infiziert.

Während der folgenden 8-wöchigen Versuchsphase wurde der Futterverbrauch pro Gruppe

täglich erfasst. In den letzten beiden Durchgängen mit H. gallinarum wurden zur Bestimmung

der täglichen Kotmenge, der Parasiteneiausscheidung pro Gramm Kot (EPG) und der Anzahl

Zusammenfassung

107

107

ausgeschiedener Parasiteneier innerhalb eines Zeitraums von 24h (Eier pro Tag, EPD) Tiere

in Einzelkäfigen gehalten.

Im A. galli Experiment wurden die Tiere zur Bestimmung der Wurmzahl pro Tier 8

Wochen nach der Infektion geschlachtet. Zum Zeitpunkt der Schlachtung erfolgte darüber

hinaus eine individuelle Kotprobenentnahme zur EPG-Bestimmung. Weiterhin wurden der

pH-Wert sowie der Gehalt flüchtiger Fettsäuren in der Blinddarm-Digesta untersucht.

Im ersten Versuch (Histomonas infizierte H. gallinarum, Kapitel 2) wurde festgestellt,

dass – unabhängig von der Fütterung - die Behandlung gegen Histomonas meleagridis sowohl

das Auftreten von H. gallinarum als auch die durchschnittliche Länge der Würmer steigert. Es

ergab sich eine Interaktion zwischen der Fütterung und der Dimetridazol-Behandlung. S-NSP

führte bei unbehandelten Tieren zu den niedrigsten Wurmzahlen, während die behandelten

Tiere die höchste Befallsintensität aufwiesen. Innerhalb aller Fütterungsvarianten hatten

behandelte Tiere jeweils höhere Wurmzahlen als unbehandelte. Die Infektion mit H.

gallinarum beeinträchtigte das Körpergewicht (BW) der Tiere, und die gleichzeitige Infektion

mit Histomonas meleagridis verstärkte diese Wirkung. Die Behandlung mit Dimetridazol

wirkte sich nicht auf die Körpergewichtsentwicklung aus. Beide NSP-Futtermischungen,

insbesondere S-NSP, führten zu geringerem Körpergewicht der Tiere.

Im Versuch mit H. gallinarum ohne Histomonas meleagridis Infektion (Kapitel 3)

steigerte die NSP-Fütterung im Vergleich zur Kontrolle sowohl die Befallhäufigkeit als auch

die Befallsstärke. Ein Einfluss des Futters auf die durchschnittliche Wurmlänge konnte

hingegen nicht beobachtet werden. Die NSP-Fütterung führte zu gesteigerter täglicher

Kotmenge. EPG, EPD und die Fruchtbarkeit der weiblichen Würmer (EPD: Anzahl

weiblicher Würmer) stiegen unter dem Einfluß von S-NSP, während I-NSP das Verhältnis

von EPG : weiblichen Würmern verringerte. Die Anzahl ausgeschiedener Parasiteneier pro

Tag stieg in der Reihenfolge CON < I-NSP < S-NSP. Sowohl die NSP-Fütterung als auch die

Infektion führten zu geringerer Körpergewichtsentwicklung. Die Infektion beeinträchtigte

zusätzlich die Futterverwertung. Die Blinddärme der mit NSP, insbesondere der mit S-NSP

gefütterten Tiere waren durchschnittlich länger als die der Kontrolltiere. Die Fütterung und

die Infektion beeinflussten das Gewicht der Blinddärme. S-NSP und die Infektion führten zu

schwereren Blinddärmen. Die Infektion steigerte sowohl das Gesamt- (inklusive Darminhalt)

als auch das Nettogewicht (ausgewaschen/gespült) der Blinddärme. Im Vergleich zu CON

und I-NSP führte S-NSP zu niedrigeren pH-Werten, zu niedrigerer Acetat- und höherer

Butyratkonzentration sowie zu größerem Pool an flüchtigen Fettsäuren (VFA) im Blinddarm.

Zusammenfassung

108

108

Die Infektion erhöhte die pH Werte und verminderte die Butyratkonzentration sowie den

VFA Pool im Blinddarm.

Im Versuch mit Ascaridia galli (Kapitel 4) erhöhte die NSP-Fütterung und

insbesondere die mit S-NSP die Befallshäufigkeit. Im Gegensatz zum Versuch mit H.

gallinarum wurde kein Einfluss auf die Wurmlänge und die Wurmfruchtbarkeit festgestellt.

Die mit A. galli infizierten Tiere wiesen unabhängig von der Fütterung, eine verminderte

Körpergewichtsentwicklung und eine ungünstigere Futterverwertung auf. Auch die NSP-

Fütterung, und wiederum vor allem S-NSP, beeinträchtigte die Körpergewichtsentwicklung

der Tiere. S-NSP bewirkte außerdem niedrigere pH Werte im Blinddarm, während die

Infektion mit Ascaridia galli diesbezüglich keinen Einfluss hatte. Die Infektion und die

Fütterung erhöhten die Mengen an VFA im Blinddarm. S-NSP steigerte die Mengen an VFA

am stärksten. Im Vergleich zu CON führte die NSP-Fütterung, insbesondere S-NSP, zur

Verdickung zur Gewichtserhöhung des Dünndarmgewebes.

Die Ergebnisse zeigen, dass H. meleagridis nicht nur den Endwirt schädigt, sondern

auch den Vektor H. gallinarum. Sowohl unlösliche als auch lösliche NSP fördern die

Etablierung einer H. gallinarum Infektion. Lösliche NSP fördern und intensivieren zusätzlich

das Auftreten von Histomonas meleagridis. Dies hat wiederum nachteilige Auswirkungen auf

die Etablierung und Entwicklung von Heterakis gallinarum. Sowohl Erbsenschalen als auch

Zichorienwurzelmehl als Quellen unlöslicher bzw. löslicher NSP fördern die Infektion mit

Histomonas–freien H. gallinarum und mit A. galli. Zichorienwurzelmehl steigert zusätzlich

die Fruchtbarkeit von H. gallinarum. Die verwendeten unlöslichen und löslichen NSP

beeinträchtigen das Wachstum der Tiere, reichern sich im Gastrointestinaltrakt an und fördern

so möglicherweise die Empfänglichkeit gegenüber Infektionen mit Nematoden.

Die Ergebnisse lassen schließen, dass Erbsenschalen und Zichorienwurzelmehl als

Quellen unlöslicher bzw. löslicher NSP ungeeignet sind, der Etablierung und Entwicklung

von Nematodeninfektionen entgegenzuwirken. Insbesondere in alternativen

Haltungssystemen, in denen Tiere einen erhöhten Infektionsdruck ausgesetzt sind und

vermehrt NSP aufnehmen (können/), muss nach anderen Bekämpfungsmöglichkeiten gesucht

werden.

Acknowledgements

109

109

ACKNOWLEDGEMENTS

First and foremost I want to thank my supervisor Prof. Dr. Dr. Mathias Gauly and my co-

supervisor Prof. Dr. Hansjörg Abel for their guidance, efforts, constructive critics and pieces

of advice that assuredly guaranteed the quality of all the works we have performed during the

study period. I am very much grateful to re-express my sincere thanks to them for both their

professional contributions and for their incredible impacts on my experiences during any of

daily conservations or at any other social event. It has been an honour to be one of their Ph.D.

students.

I am deeply thankful to Prof. Dr. Türker Savaş, from Çanakkale Onsekiz Mart

University, Turkey, not only for accepting to be the third examiner, but also for encouraging

conversations, helpful suggestions as well as for numerous supports since years in many

different ways.

I would like to extend my thanks to Prof. Dr. Gerhard Breves and Prof. Dr. Silke

Rautenschlein, from University of Veterinary Medicine Hannover, for their help. Particular

thanks are due to my colleagues Julia Humburg and Anna Schwarz, who also took part as

Ph.D. students in the same project, for their cooperation and friendship.

I am thankful to Ms. Dr. Eva Moors, Ms. Birgit Sohnrey, Mr. Erwin Tönges, Mr. Rolf

Jeromin, Mr. Jochen Köhlers, Mr. Dieter Daniel, Mr. Thomas Kraft, Mrs. Nicole Abrill and

Mr. Knut Salzmann for their kind help with various technical issues. I would like extend my

thanks to all colleagues in the Livestock Production Systems Group at the Department of

Animal Science for their help during the parasitological examinations. It was (is) a great

honour to be in the Livestock Production Science group with colleagues and friends from

different countries and cultures. I would like to thank Falko Kaufmann, Ahmad Idris, Dr. Jan

Maxa, Dr. Alexander Riek, Dr. Anas M. Abdelqader, Christian Lambertz, Dr. Morten

Friedrich, Dr. Uta König von Borstel, , Dr. Denise Völker, Christina Münch, Patricia Graf,

Phonwon Singhaphan, Dr. Amphon Warittitham, Dr. Chakrapong Chaikong, Pilasrak

Panprasert, Kalyakorn Wongrak, Katharina Tiersch, Abdussamad Muhammad Abdussamad,

Mohammed Saleh, Mazhar Shahin, Diya AI-Ramamneh, Stephan Schulze Mönking, Hanno

Hirch and Liane Lühmann for their contributions and friendships.

Prof. Dr. Wolfgang Holtz, PD Dr. Sven König, Dr. Reza Sharifi, Mrs. Ute Döring, Mr.

Burchhard Möllers, Mrs. Edelgard Dorstewitz, Mrs. Grete Thinggaard ter Meulen are

gratefully acknowledged for their kind supports.

Acknowledgements

110

110

Financial supports of the German Research Foundation (DFG) and the H. Wilhelm

Schaumann Foundation are gratefully acknowledged. I would like to thank Prof. Gauly once

more for the financial supports that he provided when the scholarships lacked during any of

the whole study period. I gratefully acknowledge my employer, Çanakkale Onsekiz Mart

University, Turkey, for the permission that allowed me to complete my Ph.D. studies in

Germany. I would like to extend my sincere thanks to the academic staff of the Department of

Animal Science at Canakkale Onsekiz Mart University for their supports. Many thanks to

Prof. Dr. İ. Yaman Yurtman, Assoc. Prof. Dr. Aynur Konyalı, Prof. Dr. Kemal Celik, Prof.

Dr. Cengiz Ataşoğlu, Mr. İ. Erbil Ersoy, Dr. Cemil Tölü, Ms. Hande Işıl Akbağ, Dr. Baver

Coşkun and Mr. Coşkun Konyalı for their supports.

I thank my wife Hatice and my daughters Melike and Su Zelal for their love,

incredible patience, support and encouragement. I am proud of you girls; you did perform

great efforts to cope with a new environment starting from almost zero since I have been

studying. I am deeply thankful to my father, brothers and sisters for continued moral support.

All those whose names are inadvertently left out, please accept my sincere gratitude.

Thank you.

Gürbüz Daş

November 2010

http://akademik.comu.edu.tr/onizle.php?cvno=A-1551�





Curriculum Vitae

111

111

Curriculum Vitae

Name : Gürbüz Daş

Date and place of birth : 04.04.1977, Göle, Turkey

E-mail : [email protected]; [email protected]

Graduate studies

1995-1997 : Animal Breeding and Health Programme,

(Assoc. Degree) Kars College of Applied Sciences of Kafkas Un., Kars, Turkey

1997-2001 (B.Sc. Degree) : Faculty of Agriculture, Department of Animal Science,

Çukurova University, Turkey

2002-2004 (M.Sc. Degree) : Graduate School of Natural and Applied Sciences, Dep. of

Animal Sci., Çanakkale Onsekiz Mart University, Turkey

2007-2010 (Ph.D.) : Ph.D. Program for Agricultural Sciences in Göttingen (PAG),

Department of Animal Sciences, Faculty of Agricultural

Sciences, Georg-August-Universität Göttingen, Germany

Professional career

2002 to present : Research Assistant position at Çanakkale Onsekiz Mart

University, Faculty of Agriculture, Department of Animal

Science, Çanakkale, Turkey

Honor and Awards

June, 1997 : Graduated at 2nd top graduate from Kars College of Applied

Sciences of Kafkas University, Turkey

June, 2001 : Graduated with the highest honor from Department of Animal

Science, Faculty of Agriculture, Cukurova University, Adana,

Turkey

May, 2004 : Awarded by the Alfred Toepfer Stiftung F.V.S. with one of

Liebig research scholarships 2004, connected with the “Justus

von Liebig Prize” at Kiel Christian-Albrechts University, Kiel,

Germany

Göttingen, 09.09.2010

mailto:[email protected]�

mailto:[email protected]�

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